| |
| |
| |
| |
| |
| |
| |
| """Bio.SeqIO support for the FASTQ and QUAL file formats. |
| |
| Note that you are expected to use this code via the Bio.SeqIO interface, as |
| shown below. |
| |
| The FASTQ file format is used frequently at the Wellcome Trust Sanger Institute |
| to bundle a FASTA sequence and its PHRED quality data (integers between 0 and |
| 90). Rather than using a single FASTQ file, often paired FASTA and QUAL files |
| are used containing the sequence and the quality information separately. |
| |
| The PHRED software reads DNA sequencing trace files, calls bases, and |
| assigns a non-negative quality value to each called base using a logged |
| transformation of the error probability, Q = -10 log10( Pe ), for example:: |
| |
| Pe = 1.0, Q = 0 |
| Pe = 0.1, Q = 10 |
| Pe = 0.01, Q = 20 |
| ... |
| Pe = 0.00000001, Q = 80 |
| Pe = 0.000000001, Q = 90 |
| |
| In typical raw sequence reads, the PHRED quality valuea will be from 0 to 40. |
| In the QUAL format these quality values are held as space separated text in |
| a FASTA like file format. In the FASTQ format, each quality values is encoded |
| with a single ASCI character using chr(Q+33), meaning zero maps to the |
| character "!" and for example 80 maps to "q". For the Sanger FASTQ standard |
| the allowed range of PHRED scores is 0 to 93 inclusive. The sequences and |
| quality are then stored in pairs in a FASTA like format. |
| |
| Unfortunately there is no official document describing the FASTQ file format, |
| and worse, several related but different variants exist. For more details, |
| please read this open access publication:: |
| |
| The Sanger FASTQ file format for sequences with quality scores, and the |
| Solexa/Illumina FASTQ variants. |
| P.J.A.Cock (Biopython), C.J.Fields (BioPerl), N.Goto (BioRuby), |
| M.L.Heuer (BioJava) and P.M. Rice (EMBOSS). |
| Nucleic Acids Research 2010 38(6):1767-1771 |
| https://doi.org/10.1093/nar/gkp1137 |
| |
| The good news is that Roche 454 sequencers can output files in the QUAL format, |
| and sensibly they use PHREP style scores like Sanger. Converting a pair of |
| FASTA and QUAL files into a Sanger style FASTQ file is easy. To extract QUAL |
| files from a Roche 454 SFF binary file, use the Roche off instrument command |
| line tool "sffinfo" with the -q or -qual argument. You can extract a matching |
| FASTA file using the -s or -seq argument instead. |
| |
| The bad news is that Solexa/Illumina did things differently - they have their |
| own scoring system AND their own incompatible versions of the FASTQ format. |
| Solexa/Illumina quality scores use Q = - 10 log10 ( Pe / (1-Pe) ), which can |
| be negative. PHRED scores and Solexa scores are NOT interchangeable (but a |
| reasonable mapping can be achieved between them, and they are approximately |
| equal for higher quality reads). |
| |
| Confusingly early Solexa pipelines produced a FASTQ like file but using their |
| own score mapping and an ASCII offset of 64. To make things worse, for the |
| Solexa/Illumina pipeline 1.3 onwards, they introduced a third variant of the |
| FASTQ file format, this time using PHRED scores (which is more consistent) but |
| with an ASCII offset of 64. |
| |
| i.e. There are at least THREE different and INCOMPATIBLE variants of the FASTQ |
| file format: The original Sanger PHRED standard, and two from Solexa/Illumina. |
| |
| The good news is that as of CASAVA version 1.8, Illumina sequencers will |
| produce FASTQ files using the standard Sanger encoding. |
| |
| You are expected to use this module via the Bio.SeqIO functions, with the |
| following format names: |
| |
| - "qual" means simple quality files using PHRED scores (e.g. from Roche 454) |
| - "fastq" means Sanger style FASTQ files using PHRED scores and an ASCII |
| offset of 33 (e.g. from the NCBI Short Read Archive and Illumina 1.8+). |
| These can potentially hold PHRED scores from 0 to 93. |
| - "fastq-sanger" is an alias for "fastq". |
| - "fastq-solexa" means old Solexa (and also very early Illumina) style FASTQ |
| files, using Solexa scores with an ASCII offset 64. These can hold Solexa |
| scores from -5 to 62. |
| - "fastq-illumina" means newer Illumina 1.3 to 1.7 style FASTQ files, using |
| PHRED scores but with an ASCII offset 64, allowing PHRED scores from 0 |
| to 62. |
| |
| We could potentially add support for "qual-solexa" meaning QUAL files which |
| contain Solexa scores, but thus far there isn't any reason to use such files. |
| |
| For example, consider the following short FASTQ file:: |
| |
| @EAS54_6_R1_2_1_413_324 |
| CCCTTCTTGTCTTCAGCGTTTCTCC |
| + |
| ;;3;;;;;;;;;;;;7;;;;;;;88 |
| @EAS54_6_R1_2_1_540_792 |
| TTGGCAGGCCAAGGCCGATGGATCA |
| + |
| ;;;;;;;;;;;7;;;;;-;;;3;83 |
| @EAS54_6_R1_2_1_443_348 |
| GTTGCTTCTGGCGTGGGTGGGGGGG |
| + |
| ;;;;;;;;;;;9;7;;.7;393333 |
| |
| This contains three reads of length 25. From the read length these were |
| probably originally from an early Solexa/Illumina sequencer but this file |
| follows the Sanger FASTQ convention (PHRED style qualities with an ASCII |
| offset of 33). This means we can parse this file using Bio.SeqIO using |
| "fastq" as the format name: |
| |
| >>> from Bio import SeqIO |
| >>> for record in SeqIO.parse("Quality/example.fastq", "fastq"): |
| ... print("%s %s" % (record.id, record.seq)) |
| EAS54_6_R1_2_1_413_324 CCCTTCTTGTCTTCAGCGTTTCTCC |
| EAS54_6_R1_2_1_540_792 TTGGCAGGCCAAGGCCGATGGATCA |
| EAS54_6_R1_2_1_443_348 GTTGCTTCTGGCGTGGGTGGGGGGG |
| |
| The qualities are held as a list of integers in each record's annotation: |
| |
| >>> print(record) |
| ID: EAS54_6_R1_2_1_443_348 |
| Name: EAS54_6_R1_2_1_443_348 |
| Description: EAS54_6_R1_2_1_443_348 |
| Number of features: 0 |
| Per letter annotation for: phred_quality |
| Seq('GTTGCTTCTGGCGTGGGTGGGGGGG') |
| >>> print(record.letter_annotations["phred_quality"]) |
| [26, 26, 26, 26, 26, 26, 26, 26, 26, 26, 26, 24, 26, 22, 26, 26, 13, 22, 26, 18, 24, 18, 18, 18, 18] |
| |
| You can use the SeqRecord format method to show this in the QUAL format: |
| |
| >>> print(record.format("qual")) |
| >EAS54_6_R1_2_1_443_348 |
| 26 26 26 26 26 26 26 26 26 26 26 24 26 22 26 26 13 22 26 18 |
| 24 18 18 18 18 |
| <BLANKLINE> |
| |
| Or go back to the FASTQ format, use "fastq" (or "fastq-sanger"): |
| |
| >>> print(record.format("fastq")) |
| @EAS54_6_R1_2_1_443_348 |
| GTTGCTTCTGGCGTGGGTGGGGGGG |
| + |
| ;;;;;;;;;;;9;7;;.7;393333 |
| <BLANKLINE> |
| |
| Or, using the Illumina 1.3+ FASTQ encoding (PHRED values with an ASCII offset |
| of 64): |
| |
| >>> print(record.format("fastq-illumina")) |
| @EAS54_6_R1_2_1_443_348 |
| GTTGCTTCTGGCGTGGGTGGGGGGG |
| + |
| ZZZZZZZZZZZXZVZZMVZRXRRRR |
| <BLANKLINE> |
| |
| You can also get Biopython to convert the scores and show a Solexa style |
| FASTQ file: |
| |
| >>> print(record.format("fastq-solexa")) |
| @EAS54_6_R1_2_1_443_348 |
| GTTGCTTCTGGCGTGGGTGGGGGGG |
| + |
| ZZZZZZZZZZZXZVZZMVZRXRRRR |
| <BLANKLINE> |
| |
| Notice that this is actually the same output as above using "fastq-illumina" |
| as the format! The reason for this is all these scores are high enough that |
| the PHRED and Solexa scores are almost equal. The differences become apparent |
| for poor quality reads. See the functions solexa_quality_from_phred and |
| phred_quality_from_solexa for more details. |
| |
| If you wanted to trim your sequences (perhaps to remove low quality regions, |
| or to remove a primer sequence), try slicing the SeqRecord objects. e.g. |
| |
| >>> sub_rec = record[5:15] |
| >>> print(sub_rec) |
| ID: EAS54_6_R1_2_1_443_348 |
| Name: EAS54_6_R1_2_1_443_348 |
| Description: EAS54_6_R1_2_1_443_348 |
| Number of features: 0 |
| Per letter annotation for: phred_quality |
| Seq('TTCTGGCGTG') |
| >>> print(sub_rec.letter_annotations["phred_quality"]) |
| [26, 26, 26, 26, 26, 26, 24, 26, 22, 26] |
| >>> print(sub_rec.format("fastq")) |
| @EAS54_6_R1_2_1_443_348 |
| TTCTGGCGTG |
| + |
| ;;;;;;9;7; |
| <BLANKLINE> |
| |
| If you wanted to, you could read in this FASTQ file, and save it as a QUAL file: |
| |
| >>> from Bio import SeqIO |
| >>> record_iterator = SeqIO.parse("Quality/example.fastq", "fastq") |
| >>> with open("Quality/temp.qual", "w") as out_handle: |
| ... SeqIO.write(record_iterator, out_handle, "qual") |
| 3 |
| |
| You can of course read in a QUAL file, such as the one we just created: |
| |
| >>> from Bio import SeqIO |
| >>> for record in SeqIO.parse("Quality/temp.qual", "qual"): |
| ... print("%s read of length %d" % (record.id, len(record.seq))) |
| EAS54_6_R1_2_1_413_324 read of length 25 |
| EAS54_6_R1_2_1_540_792 read of length 25 |
| EAS54_6_R1_2_1_443_348 read of length 25 |
| |
| Notice that QUAL files don't have a proper sequence present! But the quality |
| information is there: |
| |
| >>> print(record) |
| ID: EAS54_6_R1_2_1_443_348 |
| Name: EAS54_6_R1_2_1_443_348 |
| Description: EAS54_6_R1_2_1_443_348 |
| Number of features: 0 |
| Per letter annotation for: phred_quality |
| Undefined sequence of length 25 |
| >>> print(record.letter_annotations["phred_quality"]) |
| [26, 26, 26, 26, 26, 26, 26, 26, 26, 26, 26, 24, 26, 22, 26, 26, 13, 22, 26, 18, 24, 18, 18, 18, 18] |
| |
| Just to keep things tidy, if you are following this example yourself, you can |
| delete this temporary file now: |
| |
| >>> import os |
| >>> os.remove("Quality/temp.qual") |
| |
| Sometimes you won't have a FASTQ file, but rather just a pair of FASTA and QUAL |
| files. Because the Bio.SeqIO system is designed for reading single files, you |
| would have to read the two in separately and then combine the data. However, |
| since this is such a common thing to want to do, there is a helper iterator |
| defined in this module that does this for you - PairedFastaQualIterator. |
| |
| Alternatively, if you have enough RAM to hold all the records in memory at once, |
| then a simple dictionary approach would work: |
| |
| >>> from Bio import SeqIO |
| >>> reads = SeqIO.to_dict(SeqIO.parse("Quality/example.fasta", "fasta")) |
| >>> for rec in SeqIO.parse("Quality/example.qual", "qual"): |
| ... reads[rec.id].letter_annotations["phred_quality"]=rec.letter_annotations["phred_quality"] |
| |
| You can then access any record by its key, and get both the sequence and the |
| quality scores. |
| |
| >>> print(reads["EAS54_6_R1_2_1_540_792"].format("fastq")) |
| @EAS54_6_R1_2_1_540_792 |
| TTGGCAGGCCAAGGCCGATGGATCA |
| + |
| ;;;;;;;;;;;7;;;;;-;;;3;83 |
| <BLANKLINE> |
| |
| It is important that you explicitly tell Bio.SeqIO which FASTQ variant you are |
| using ("fastq" or "fastq-sanger" for the Sanger standard using PHRED values, |
| "fastq-solexa" for the original Solexa/Illumina variant, or "fastq-illumina" |
| for the more recent variant), as this cannot be detected reliably |
| automatically. |
| |
| To illustrate this problem, let's consider an artificial example: |
| |
| >>> from Bio.Seq import Seq |
| >>> from Bio.SeqRecord import SeqRecord |
| >>> test = SeqRecord(Seq("NACGTACGTA"), id="Test", description="Made up!") |
| >>> print(test.format("fasta")) |
| >Test Made up! |
| NACGTACGTA |
| <BLANKLINE> |
| >>> print(test.format("fastq")) |
| Traceback (most recent call last): |
| ... |
| ValueError: No suitable quality scores found in letter_annotations of SeqRecord (id=Test). |
| |
| We created a sample SeqRecord, and can show it in FASTA format - but for QUAL |
| or FASTQ format we need to provide some quality scores. These are held as a |
| list of integers (one for each base) in the letter_annotations dictionary: |
| |
| >>> test.letter_annotations["phred_quality"] = [0, 1, 2, 3, 4, 5, 10, 20, 30, 40] |
| >>> print(test.format("qual")) |
| >Test Made up! |
| 0 1 2 3 4 5 10 20 30 40 |
| <BLANKLINE> |
| >>> print(test.format("fastq")) |
| @Test Made up! |
| NACGTACGTA |
| + |
| !"#$%&+5?I |
| <BLANKLINE> |
| |
| We can check this FASTQ encoding - the first PHRED quality was zero, and this |
| mapped to a exclamation mark, while the final score was 40 and this mapped to |
| the letter "I": |
| |
| >>> ord('!') - 33 |
| 0 |
| >>> ord('I') - 33 |
| 40 |
| >>> [ord(letter)-33 for letter in '!"#$%&+5?I'] |
| [0, 1, 2, 3, 4, 5, 10, 20, 30, 40] |
| |
| Similarly, we could produce an Illumina 1.3 to 1.7 style FASTQ file using PHRED |
| scores with an offset of 64: |
| |
| >>> print(test.format("fastq-illumina")) |
| @Test Made up! |
| NACGTACGTA |
| + |
| @ABCDEJT^h |
| <BLANKLINE> |
| |
| And we can check this too - the first PHRED score was zero, and this mapped to |
| "@", while the final score was 40 and this mapped to "h": |
| |
| >>> ord("@") - 64 |
| 0 |
| >>> ord("h") - 64 |
| 40 |
| >>> [ord(letter)-64 for letter in "@ABCDEJT^h"] |
| [0, 1, 2, 3, 4, 5, 10, 20, 30, 40] |
| |
| Notice how different the standard Sanger FASTQ and the Illumina 1.3 to 1.7 style |
| FASTQ files look for the same data! Then we have the older Solexa/Illumina |
| format to consider which encodes Solexa scores instead of PHRED scores. |
| |
| First let's see what Biopython says if we convert the PHRED scores into Solexa |
| scores (rounding to one decimal place): |
| |
| >>> for q in [0, 1, 2, 3, 4, 5, 10, 20, 30, 40]: |
| ... print("PHRED %i maps to Solexa %0.1f" % (q, solexa_quality_from_phred(q))) |
| PHRED 0 maps to Solexa -5.0 |
| PHRED 1 maps to Solexa -5.0 |
| PHRED 2 maps to Solexa -2.3 |
| PHRED 3 maps to Solexa -0.0 |
| PHRED 4 maps to Solexa 1.8 |
| PHRED 5 maps to Solexa 3.3 |
| PHRED 10 maps to Solexa 9.5 |
| PHRED 20 maps to Solexa 20.0 |
| PHRED 30 maps to Solexa 30.0 |
| PHRED 40 maps to Solexa 40.0 |
| |
| Now here is the record using the old Solexa style FASTQ file: |
| |
| >>> print(test.format("fastq-solexa")) |
| @Test Made up! |
| NACGTACGTA |
| + |
| ;;>@BCJT^h |
| <BLANKLINE> |
| |
| Again, this is using an ASCII offset of 64, so we can check the Solexa scores: |
| |
| >>> [ord(letter)-64 for letter in ";;>@BCJT^h"] |
| [-5, -5, -2, 0, 2, 3, 10, 20, 30, 40] |
| |
| This explains why the last few letters of this FASTQ output matched that using |
| the Illumina 1.3 to 1.7 format - high quality PHRED scores and Solexa scores |
| are approximately equal. |
| |
| """ |
|
|
| import warnings |
| from math import log |
| from abc import abstractmethod |
| from typing import Any |
| from collections.abc import Callable |
| from typing import IO |
| from collections.abc import Iterator |
| from collections.abc import Mapping |
| from typing import Optional |
| from collections.abc import Sequence |
| from typing import Union |
| from collections.abc import Iterable |
| import array |
| from dataclasses import dataclass |
|
|
| from Bio import BiopythonParserWarning |
| from Bio import BiopythonWarning |
| from Bio import BiopythonDeprecationWarning |
| from Bio import StreamModeError |
| from Bio.File import as_handle |
| from Bio.Seq import Seq |
| from Bio.SeqRecord import SeqRecord |
|
|
| from .Interfaces import _clean |
| from .Interfaces import _get_seq_string |
| from .Interfaces import _TextIOSource |
| from .Interfaces import SequenceIterator |
| from .Interfaces import SequenceWriter |
|
|
| |
| |
| SANGER_SCORE_OFFSET = 33 |
| SOLEXA_SCORE_OFFSET = 64 |
| INVALID_CHAR_CODE = 200 |
| INVALID_CHAR = bytes((INVALID_CHAR_CODE,)) |
|
|
|
|
| def solexa_quality_from_phred(phred_quality: float) -> float: |
| """Convert a PHRED quality (range 0 to about 90) to a Solexa quality. |
| |
| PHRED and Solexa quality scores are both log transformations of a |
| probality of error (high score = low probability of error). This function |
| takes a PHRED score, transforms it back to a probability of error, and |
| then re-expresses it as a Solexa score. This assumes the error estimates |
| are equivalent. |
| |
| How does this work exactly? Well the PHRED quality is minus ten times the |
| base ten logarithm of the probability of error:: |
| |
| phred_quality = -10*log(error,10) |
| |
| Therefore, turning this round:: |
| |
| error = 10 ** (- phred_quality / 10) |
| |
| Now, Solexa qualities use a different log transformation:: |
| |
| solexa_quality = -10*log(error/(1-error),10) |
| |
| After substitution and a little manipulation we get:: |
| |
| solexa_quality = 10*log(10**(phred_quality/10.0) - 1, 10) |
| |
| However, real Solexa files use a minimum quality of -5. This does have a |
| good reason - a random base call would be correct 25% of the time, |
| and thus have a probability of error of 0.75, which gives 1.25 as the PHRED |
| quality, or -4.77 as the Solexa quality. Thus (after rounding), a random |
| nucleotide read would have a PHRED quality of 1, or a Solexa quality of -5. |
| |
| Taken literally, this logarithic formula would map a PHRED quality of zero |
| to a Solexa quality of minus infinity. Of course, taken literally, a PHRED |
| score of zero means a probability of error of one (i.e. the base call is |
| definitely wrong), which is worse than random! In practice, a PHRED quality |
| of zero usually means a default value, or perhaps random - and therefore |
| mapping it to the minimum Solexa score of -5 is reasonable. |
| |
| In conclusion, we follow EMBOSS, and take this logarithmic formula but also |
| apply a minimum value of -5.0 for the Solexa quality, and also map a PHRED |
| quality of zero to -5.0 as well. |
| |
| Note this function will return a floating point number, it is up to you to |
| round this to the nearest integer if appropriate. e.g. |
| |
| >>> print("%0.2f" % round(solexa_quality_from_phred(80), 2)) |
| 80.00 |
| >>> print("%0.2f" % round(solexa_quality_from_phred(50), 2)) |
| 50.00 |
| >>> print("%0.2f" % round(solexa_quality_from_phred(20), 2)) |
| 19.96 |
| >>> print("%0.2f" % round(solexa_quality_from_phred(10), 2)) |
| 9.54 |
| >>> print("%0.2f" % round(solexa_quality_from_phred(5), 2)) |
| 3.35 |
| >>> print("%0.2f" % round(solexa_quality_from_phred(4), 2)) |
| 1.80 |
| >>> print("%0.2f" % round(solexa_quality_from_phred(3), 2)) |
| -0.02 |
| >>> print("%0.2f" % round(solexa_quality_from_phred(2), 2)) |
| -2.33 |
| >>> print("%0.2f" % round(solexa_quality_from_phred(1), 2)) |
| -5.00 |
| >>> print("%0.2f" % round(solexa_quality_from_phred(0), 2)) |
| -5.00 |
| |
| Notice that for high quality reads PHRED and Solexa scores are numerically |
| equal. The differences are important for poor quality reads, where PHRED |
| has a minimum of zero but Solexa scores can be negative. |
| |
| Finally, as a special case where None is used for a "missing value", None |
| is returned: |
| |
| >>> print(solexa_quality_from_phred(None)) |
| None |
| """ |
| if phred_quality is None: |
| |
| |
| return None |
| elif phred_quality > 0: |
| |
| |
| return max(-5.0, 10 * log(10 ** (phred_quality / 10.0) - 1, 10)) |
| elif phred_quality == 0: |
| |
| return -5.0 |
| else: |
| raise ValueError( |
| f"PHRED qualities must be positive (or zero), not {phred_quality!r}" |
| ) |
|
|
|
|
| def phred_quality_from_solexa(solexa_quality: float) -> float: |
| """Convert a Solexa quality (which can be negative) to a PHRED quality. |
| |
| PHRED and Solexa quality scores are both log transformations of a |
| probality of error (high score = low probability of error). This function |
| takes a Solexa score, transforms it back to a probability of error, and |
| then re-expresses it as a PHRED score. This assumes the error estimates |
| are equivalent. |
| |
| The underlying formulas are given in the documentation for the sister |
| function solexa_quality_from_phred, in this case the operation is:: |
| |
| phred_quality = 10*log(10**(solexa_quality/10.0) + 1, 10) |
| |
| This will return a floating point number, it is up to you to round this to |
| the nearest integer if appropriate. e.g. |
| |
| >>> print("%0.2f" % round(phred_quality_from_solexa(80), 2)) |
| 80.00 |
| >>> print("%0.2f" % round(phred_quality_from_solexa(20), 2)) |
| 20.04 |
| >>> print("%0.2f" % round(phred_quality_from_solexa(10), 2)) |
| 10.41 |
| >>> print("%0.2f" % round(phred_quality_from_solexa(0), 2)) |
| 3.01 |
| >>> print("%0.2f" % round(phred_quality_from_solexa(-5), 2)) |
| 1.19 |
| |
| Note that a solexa_quality less then -5 is not expected, will trigger a |
| warning, but will still be converted as per the logarithmic mapping |
| (giving a number between 0 and 1.19 back). |
| |
| As a special case where None is used for a "missing value", None is |
| returned: |
| |
| >>> print(phred_quality_from_solexa(None)) |
| None |
| """ |
| if solexa_quality is None: |
| |
| return None |
| if solexa_quality < -5: |
| warnings.warn( |
| f"Solexa quality less than -5 passed, {solexa_quality!r}", BiopythonWarning |
| ) |
| return 10 * log(10 ** (solexa_quality / 10.0) + 1, 10) |
|
|
|
|
| def _get_phred_quality(record: SeqRecord) -> list[float] | list[int]: |
| """Extract PHRED qualities from a SeqRecord's letter_annotations (PRIVATE). |
| |
| If there are no PHRED qualities, but there are Solexa qualities, those are |
| used instead after conversion. |
| """ |
| try: |
| return record.letter_annotations["phred_quality"] |
| except KeyError: |
| pass |
| try: |
| return [ |
| phred_quality_from_solexa(q) |
| for q in record.letter_annotations["solexa_quality"] |
| ] |
| except KeyError: |
| raise ValueError( |
| "No suitable quality scores found in " |
| "letter_annotations of SeqRecord (id=%s)." % record.id |
| ) from None |
|
|
|
|
| |
| _phred_to_sanger_quality_str = { |
| qp: chr(min(126, qp + SANGER_SCORE_OFFSET)) for qp in range(93 + 1) |
| } |
| |
| _solexa_to_sanger_quality_str = { |
| qs: chr(min(126, int(round(phred_quality_from_solexa(qs)) + SANGER_SCORE_OFFSET))) |
| for qs in range(-5, 93 + 1) |
| } |
|
|
|
|
| def _get_sanger_quality_str(record: SeqRecord) -> str: |
| """Return a Sanger FASTQ encoded quality string (PRIVATE). |
| |
| >>> from Bio.Seq import Seq |
| >>> from Bio.SeqRecord import SeqRecord |
| >>> r = SeqRecord(Seq("ACGTAN"), id="Test", |
| ... letter_annotations = {"phred_quality":[50, 40, 30, 20, 10, 0]}) |
| >>> _get_sanger_quality_str(r) |
| 'SI?5+!' |
| |
| If as in the above example (or indeed a SeqRecord parser with Bio.SeqIO), |
| the PHRED qualities are integers, this function is able to use a very fast |
| pre-cached mapping. However, if they are floats which differ slightly, then |
| it has to do the appropriate rounding - which is slower: |
| |
| >>> r2 = SeqRecord(Seq("ACGTAN"), id="Test2", |
| ... letter_annotations = {"phred_quality":[50.0, 40.05, 29.99, 20, 9.55, 0.01]}) |
| >>> _get_sanger_quality_str(r2) |
| 'SI?5+!' |
| |
| If your scores include a None value, this raises an exception: |
| |
| >>> r3 = SeqRecord(Seq("ACGTAN"), id="Test3", |
| ... letter_annotations = {"phred_quality":[50, 40, 30, 20, 10, None]}) |
| >>> _get_sanger_quality_str(r3) |
| Traceback (most recent call last): |
| ... |
| TypeError: A quality value of None was found |
| |
| If (strangely) your record has both PHRED and Solexa scores, then the PHRED |
| scores are used in preference: |
| |
| >>> r4 = SeqRecord(Seq("ACGTAN"), id="Test4", |
| ... letter_annotations = {"phred_quality":[50, 40, 30, 20, 10, 0], |
| ... "solexa_quality":[-5, -4, 0, None, 0, 40]}) |
| >>> _get_sanger_quality_str(r4) |
| 'SI?5+!' |
| |
| If there are no PHRED scores, but there are Solexa scores, these are used |
| instead (after the appropriate conversion): |
| |
| >>> r5 = SeqRecord(Seq("ACGTAN"), id="Test5", |
| ... letter_annotations = {"solexa_quality":[40, 30, 20, 10, 0, -5]}) |
| >>> _get_sanger_quality_str(r5) |
| 'I?5+$"' |
| |
| Again, integer Solexa scores can be looked up in a pre-cached mapping making |
| this very fast. You can still use approximate floating point scores: |
| |
| >>> r6 = SeqRecord(Seq("ACGTAN"), id="Test6", |
| ... letter_annotations = {"solexa_quality":[40.1, 29.7, 20.01, 10, 0.0, -4.9]}) |
| >>> _get_sanger_quality_str(r6) |
| 'I?5+$"' |
| |
| Notice that due to the limited range of printable ASCII characters, a |
| PHRED quality of 93 is the maximum that can be held in an Illumina FASTQ |
| file (using ASCII 126, the tilde). This function will issue a warning |
| in this situation. |
| """ |
| |
| |
| |
| try: |
| |
| qualities = record.letter_annotations["phred_quality"] |
| except KeyError: |
| |
| pass |
| else: |
| |
| try: |
| return "".join(_phred_to_sanger_quality_str[qp] for qp in qualities) |
| except KeyError: |
| |
| pass |
| if None in qualities: |
| raise TypeError("A quality value of None was found") |
| if max(qualities) >= 93.5: |
| warnings.warn( |
| "Data loss - max PHRED quality 93 in Sanger FASTQ", BiopythonWarning |
| ) |
| |
| return "".join( |
| chr(min(126, int(round(qp)) + SANGER_SCORE_OFFSET)) for qp in qualities |
| ) |
| |
| try: |
| qualities = record.letter_annotations["solexa_quality"] |
| except KeyError: |
| raise ValueError( |
| "No suitable quality scores found in " |
| "letter_annotations of SeqRecord (id=%s)." % record.id |
| ) from None |
| |
| try: |
| return "".join(_solexa_to_sanger_quality_str[qs] for qs in qualities) |
| except KeyError: |
| |
| pass |
| if None in qualities: |
| raise TypeError("A quality value of None was found") |
| |
| |
| if max(qualities) >= 93.5: |
| warnings.warn( |
| "Data loss - max PHRED quality 93 in Sanger FASTQ", BiopythonWarning |
| ) |
| |
| return "".join( |
| chr(min(126, int(round(phred_quality_from_solexa(qs))) + SANGER_SCORE_OFFSET)) |
| for qs in qualities |
| ) |
|
|
|
|
| |
| assert 62 + SOLEXA_SCORE_OFFSET == 126 |
| _phred_to_illumina_quality_str = { |
| qp: chr(qp + SOLEXA_SCORE_OFFSET) for qp in range(62 + 1) |
| } |
| |
| _solexa_to_illumina_quality_str = { |
| qs: chr(int(round(phred_quality_from_solexa(qs))) + SOLEXA_SCORE_OFFSET) |
| for qs in range(-5, 62 + 1) |
| } |
|
|
|
|
| def _get_illumina_quality_str(record: SeqRecord) -> str: |
| """Return an Illumina 1.3 to 1.7 FASTQ encoded quality string (PRIVATE). |
| |
| Notice that due to the limited range of printable ASCII characters, a |
| PHRED quality of 62 is the maximum that can be held in an Illumina FASTQ |
| file (using ASCII 126, the tilde). This function will issue a warning |
| in this situation. |
| """ |
| |
| |
| |
| try: |
| |
| qualities = record.letter_annotations["phred_quality"] |
| except KeyError: |
| |
| pass |
| else: |
| |
| try: |
| return "".join(_phred_to_illumina_quality_str[qp] for qp in qualities) |
| except KeyError: |
| |
| pass |
| if None in qualities: |
| raise TypeError("A quality value of None was found") |
| if max(qualities) >= 62.5: |
| warnings.warn( |
| "Data loss - max PHRED quality 62 in Illumina FASTQ", BiopythonWarning |
| ) |
| |
| return "".join( |
| chr(min(126, int(round(qp)) + SOLEXA_SCORE_OFFSET)) for qp in qualities |
| ) |
| |
| try: |
| qualities = record.letter_annotations["solexa_quality"] |
| except KeyError: |
| raise ValueError( |
| "No suitable quality scores found in " |
| "letter_annotations of SeqRecord (id=%s)." % record.id |
| ) from None |
| |
| try: |
| return "".join(_solexa_to_illumina_quality_str[qs] for qs in qualities) |
| except KeyError: |
| |
| pass |
| if None in qualities: |
| raise TypeError("A quality value of None was found") |
| |
| |
| if max(qualities) >= 62.5: |
| warnings.warn( |
| "Data loss - max PHRED quality 62 in Illumina FASTQ", BiopythonWarning |
| ) |
| |
| return "".join( |
| chr(min(126, int(round(phred_quality_from_solexa(qs))) + SOLEXA_SCORE_OFFSET)) |
| for qs in qualities |
| ) |
|
|
|
|
| |
| assert 62 + SOLEXA_SCORE_OFFSET == 126 |
| _solexa_to_solexa_quality_str = { |
| qs: chr(min(126, qs + SOLEXA_SCORE_OFFSET)) for qs in range(-5, 62 + 1) |
| } |
| |
| _phred_to_solexa_quality_str = { |
| qp: chr(min(126, int(round(solexa_quality_from_phred(qp))) + SOLEXA_SCORE_OFFSET)) |
| for qp in range(62 + 1) |
| } |
|
|
|
|
| def _get_solexa_quality_str(record: SeqRecord) -> str: |
| """Return a Solexa FASTQ encoded quality string (PRIVATE). |
| |
| Notice that due to the limited range of printable ASCII characters, a |
| Solexa quality of 62 is the maximum that can be held in a Solexa FASTQ |
| file (using ASCII 126, the tilde). This function will issue a warning |
| in this situation. |
| """ |
| |
| |
| |
| try: |
| |
| qualities = record.letter_annotations["solexa_quality"] |
| except KeyError: |
| |
| pass |
| else: |
| |
| try: |
| return "".join(_solexa_to_solexa_quality_str[qs] for qs in qualities) |
| except KeyError: |
| |
| pass |
| if None in qualities: |
| raise TypeError("A quality value of None was found") |
| if max(qualities) >= 62.5: |
| warnings.warn( |
| "Data loss - max Solexa quality 62 in Solexa FASTQ", BiopythonWarning |
| ) |
| |
| return "".join( |
| chr(min(126, int(round(qs)) + SOLEXA_SCORE_OFFSET)) for qs in qualities |
| ) |
| |
| try: |
| qualities = record.letter_annotations["phred_quality"] |
| except KeyError: |
| raise ValueError( |
| "No suitable quality scores found in " |
| "letter_annotations of SeqRecord (id=%s)." % record.id |
| ) from None |
| |
| try: |
| return "".join(_phred_to_solexa_quality_str[qp] for qp in qualities) |
| except KeyError: |
| |
| |
| pass |
| if None in qualities: |
| raise TypeError("A quality value of None was found") |
| |
| |
| if max(qualities) >= 62.5: |
| warnings.warn( |
| "Data loss - max Solexa quality 62 in Solexa FASTQ", BiopythonWarning |
| ) |
| return "".join( |
| chr(min(126, int(round(solexa_quality_from_phred(qp))) + SOLEXA_SCORE_OFFSET)) |
| for qp in qualities |
| ) |
|
|
|
|
| |
| def FastqGeneralIterator(source: _TextIOSource) -> Iterator[tuple[str, str, str]]: |
| """Iterate over Fastq records as string tuples (not as SeqRecord objects). |
| |
| Arguments: |
| - source - input stream opened in text mode, or a path to a file |
| |
| This code does not try to interpret the quality string numerically. It |
| just returns tuples of the title, sequence and quality as strings. For |
| the sequence and quality, any whitespace (such as new lines) is removed. |
| |
| Our SeqRecord based FASTQ iterators call this function internally, and then |
| turn the strings into a SeqRecord objects, mapping the quality string into |
| a list of numerical scores. If you want to do a custom quality mapping, |
| then you might consider calling this function directly. |
| |
| For parsing FASTQ files, the title string from the "@" line at the start |
| of each record can optionally be omitted on the "+" lines. If it is |
| repeated, it must be identical. |
| |
| The sequence string and the quality string can optionally be split over |
| multiple lines, although several sources discourage this. In comparison, |
| for the FASTA file format line breaks between 60 and 80 characters are |
| the norm. |
| |
| **WARNING** - Because the "@" character can appear in the quality string, |
| this can cause problems as this is also the marker for the start of |
| a new sequence. In fact, the "+" sign can also appear as well. Some |
| sources recommended having no line breaks in the quality to avoid this, |
| but even that is not enough, consider this example:: |
| |
| @071113_EAS56_0053:1:1:998:236 |
| TTTCTTGCCCCCATAGACTGAGACCTTCCCTAAATA |
| +071113_EAS56_0053:1:1:998:236 |
| IIIIIIIIIIIIIIIIIIIIIIIIIIIIICII+III |
| @071113_EAS56_0053:1:1:182:712 |
| ACCCAGCTAATTTTTGTATTTTTGTTAGAGACAGTG |
| + |
| @IIIIIIIIIIIIIIICDIIIII<%<6&-*).(*%+ |
| @071113_EAS56_0053:1:1:153:10 |
| TGTTCTGAAGGAAGGTGTGCGTGCGTGTGTGTGTGT |
| + |
| IIIIIIIIIIIICIIGIIIII>IAIIIE65I=II:6 |
| @071113_EAS56_0053:1:3:990:501 |
| TGGGAGGTTTTATGTGGA |
| AAGCAGCAATGTACAAGA |
| + |
| IIIIIII.IIIIII1@44 |
| @-7.%<&+/$/%4(++(% |
| |
| This is four PHRED encoded FASTQ entries originally from an NCBI source |
| (given the read length of 36, these are probably Solexa Illumina reads where |
| the quality has been mapped onto the PHRED values). |
| |
| This example has been edited to illustrate some of the nasty things allowed |
| in the FASTQ format. Firstly, on the "+" lines most but not all of the |
| (redundant) identifiers are omitted. In real files it is likely that all or |
| none of these extra identifiers will be present. |
| |
| Secondly, while the first three sequences have been shown without line |
| breaks, the last has been split over multiple lines. In real files any line |
| breaks are likely to be consistent. |
| |
| Thirdly, some of the quality string lines start with an "@" character. For |
| the second record this is unavoidable. However for the fourth sequence this |
| only happens because its quality string is split over two lines. A naive |
| parser could wrongly treat any line starting with an "@" as the beginning of |
| a new sequence! This code copes with this possible ambiguity by keeping |
| track of the length of the sequence which gives the expected length of the |
| quality string. |
| |
| Using this tricky example file as input, this short bit of code demonstrates |
| what this parsing function would return: |
| |
| >>> with open("Quality/tricky.fastq") as handle: |
| ... for (title, sequence, quality) in FastqGeneralIterator(handle): |
| ... print(title) |
| ... print("%s %s" % (sequence, quality)) |
| ... |
| 071113_EAS56_0053:1:1:998:236 |
| TTTCTTGCCCCCATAGACTGAGACCTTCCCTAAATA IIIIIIIIIIIIIIIIIIIIIIIIIIIIICII+III |
| 071113_EAS56_0053:1:1:182:712 |
| ACCCAGCTAATTTTTGTATTTTTGTTAGAGACAGTG @IIIIIIIIIIIIIIICDIIIII<%<6&-*).(*%+ |
| 071113_EAS56_0053:1:1:153:10 |
| TGTTCTGAAGGAAGGTGTGCGTGCGTGTGTGTGTGT IIIIIIIIIIIICIIGIIIII>IAIIIE65I=II:6 |
| 071113_EAS56_0053:1:3:990:501 |
| TGGGAGGTTTTATGTGGAAAGCAGCAATGTACAAGA IIIIIII.IIIIII1@44@-7.%<&+/$/%4(++(% |
| |
| Finally we note that some sources state that the quality string should |
| start with "!" (which using the PHRED mapping means the first letter always |
| has a quality score of zero). This rather restrictive rule is not widely |
| observed, so is therefore ignored here. One plus point about this "!" rule |
| is that (provided there are no line breaks in the quality sequence) it |
| would prevent the above problem with the "@" character. |
| """ |
| with as_handle(source) as handle: |
| if handle.read(0) != "": |
| raise StreamModeError("Fastq files must be opened in text mode") from None |
|
|
| line = handle.readline() |
| if line == "": |
| return |
|
|
| while True: |
| if line[0] != "@": |
| raise ValueError( |
| "Records in Fastq files should start with '@' character" |
| ) |
| title_line = line[1:].rstrip() |
| seq_string = "" |
| |
| |
| for line in handle: |
| if line[0] == "+": |
| break |
| seq_string += line.rstrip() |
| else: |
| if seq_string: |
| raise ValueError("End of file without quality information.") |
| else: |
| raise ValueError("Unexpected end of file") |
| |
| second_title = line[1:].rstrip() |
| if second_title and second_title != title_line: |
| raise ValueError("Sequence and quality captions differ.") |
| |
| |
| if " " in seq_string or "\t" in seq_string: |
| raise ValueError("Whitespace is not allowed in the sequence.") |
| seq_len = len(seq_string) |
|
|
| |
| |
| line = None |
| quality_string = "" |
| for line in handle: |
| if line[0] == "@": |
| |
| |
| |
| |
| if len(quality_string) >= seq_len: |
| |
| |
| break |
| |
| quality_string += line.rstrip() |
| else: |
| if line is None: |
| raise ValueError("Unexpected end of file") |
| line = None |
|
|
| if seq_len != len(quality_string): |
| raise ValueError( |
| "Lengths of sequence and quality values differs for %s (%i and %i)." |
| % (title_line, seq_len, len(quality_string)) |
| ) |
|
|
| |
| yield (title_line, seq_string, quality_string) |
|
|
| if line is None: |
| break |
|
|
|
|
| class FastqIteratorAbstractBaseClass(SequenceIterator[str]): |
| """Abstract base class for FASTQ file parsers.""" |
|
|
| modes = "t" |
|
|
| @property |
| @abstractmethod |
| def q_mapping(self): |
| """Dictionary that maps letters in the quality string to quality values.""" |
| pass |
|
|
| @property |
| @abstractmethod |
| def q_key(self): |
| """Key name (string) of the quality values in record.letter_annotations.""" |
| pass |
|
|
| def __init__(self, source): |
| """Iterate over FASTQ records as SeqRecord objects. |
| |
| Arguments: |
| - source - input stream opened in text mode, or a path to a file |
| |
| The quality values are stored in the `letter_annotations` dictionary |
| attribute under the key `q_key`. |
| """ |
| super().__init__(source, fmt="Fastq") |
| self.line = None |
|
|
| def __next__(self) -> SeqRecord: |
| """Parse the file and generate SeqRecord objects.""" |
|
|
| line = self.line |
| if line is None: |
| line = self.stream.readline() |
| if not line: |
| raise StopIteration |
| if line[0] != "@": |
| raise ValueError("Records in Fastq files should start with '@' character") |
| title_line = line[1:].rstrip() |
| seq_string = "" |
| |
| |
| for line in self.stream: |
| if line[0] == "+": |
| break |
| seq_string += line.rstrip() |
| else: |
| if seq_string: |
| raise ValueError("End of file without quality information.") |
| else: |
| raise ValueError("Unexpected end of file") |
| seq_len = len(seq_string) |
| |
| second_title = line[1:].rstrip() |
| if second_title and second_title != title_line: |
| raise ValueError("Sequence and quality captions differ.") |
|
|
| |
| if not seq_string.isprintable() or " " in seq_string: |
| |
| raise ValueError("Whitespace is not allowed in the sequence.") |
|
|
| |
| |
| line = None |
| quality_string = "" |
| for line in self.stream: |
| if line[0] == "@": |
| |
| |
| |
| |
| if len(quality_string) >= seq_len: |
| |
| |
| self.line = line |
| break |
| |
| quality_string += line.rstrip() |
| else: |
| if line is None: |
| raise ValueError("Unexpected end of file") |
| self.line = None |
|
|
| descr = title_line |
| id = descr.split()[0] |
| name = id |
|
|
| if not quality_string.isascii(): |
| |
| index = _find_index_where(quality_string, lambda c: not c.isascii()) |
| assert index >= 0, "Non-ascii char in qualities not found. Biopython bug?" |
|
|
| details = "is not an ASCII character" |
| raise InvalidCharError(quality_string, index, details) |
|
|
| if len(quality_string) != seq_len: |
| |
| raise ValueError( |
| f"Lengths of sequence and quality values differs for {title_line} ({seq_len} and {len(quality_string)})." |
| ) |
|
|
| byte_scores = quality_string.encode().translate(self.q_mapping) |
|
|
| if INVALID_CHAR in byte_scores: |
| |
| invalid_index = byte_scores.find(INVALID_CHAR_CODE) |
|
|
| details = "not in correct range (are you sure you're using the right QualityIO parser?)" |
| raise InvalidCharError(quality_string, invalid_index, details) |
|
|
| |
| qualities = array.array("b", byte_scores).tolist() |
|
|
| |
| |
| record = SeqRecord._from_validated( |
| Seq(seq_string.encode()), |
| id=id, |
| name=name, |
| description=descr, |
| letter_annotations={self.q_key: qualities}, |
| ) |
| return record |
|
|
|
|
| class FastqPhredIterator(FastqIteratorAbstractBaseClass): |
| """Parser for FASTQ files.""" |
|
|
| assert SANGER_SCORE_OFFSET == ord("!") |
| |
| |
| |
| |
| |
| q_mapping = bytes( |
| ( |
| letter - SANGER_SCORE_OFFSET |
| if SANGER_SCORE_OFFSET <= letter < 94 + SANGER_SCORE_OFFSET |
| else INVALID_CHAR_CODE |
| ) |
| for letter in range(256) |
| ) |
|
|
| q_key = "phred_quality" |
|
|
| def __init__( |
| self, |
| source: _TextIOSource, |
| alphabet: None = None, |
| ): |
| """Iterate over FASTQ records as SeqRecord objects. |
| |
| Arguments: |
| - source - input stream opened in text mode, or a path to a file |
| - alphabet - optional alphabet, no longer used. Leave as None. |
| |
| For each sequence in a (Sanger style) FASTQ file there is a matching string |
| encoding the PHRED qualities (integers between 0 and about 90) using ASCII |
| values with an offset of 33. |
| |
| For example, consider a file containing three short reads:: |
| |
| @EAS54_6_R1_2_1_413_324 |
| CCCTTCTTGTCTTCAGCGTTTCTCC |
| + |
| ;;3;;;;;;;;;;;;7;;;;;;;88 |
| @EAS54_6_R1_2_1_540_792 |
| TTGGCAGGCCAAGGCCGATGGATCA |
| + |
| ;;;;;;;;;;;7;;;;;-;;;3;83 |
| @EAS54_6_R1_2_1_443_348 |
| GTTGCTTCTGGCGTGGGTGGGGGGG |
| + |
| ;;;;;;;;;;;9;7;;.7;393333 |
| |
| For each sequence (e.g. "CCCTTCTTGTCTTCAGCGTTTCTCC") there is a matching |
| string encoding the PHRED qualities using a ASCII values with an offset of |
| 33 (e.g. ";;3;;;;;;;;;;;;7;;;;;;;88"). |
| |
| Using this module directly you might run: |
| |
| >>> with open("Quality/example.fastq") as handle: |
| ... for record in FastqPhredIterator(handle): |
| ... print("%s %s" % (record.id, record.seq)) |
| EAS54_6_R1_2_1_413_324 CCCTTCTTGTCTTCAGCGTTTCTCC |
| EAS54_6_R1_2_1_540_792 TTGGCAGGCCAAGGCCGATGGATCA |
| EAS54_6_R1_2_1_443_348 GTTGCTTCTGGCGTGGGTGGGGGGG |
| |
| Typically however, you would call this via Bio.SeqIO instead with "fastq" |
| (or "fastq-sanger") as the format: |
| |
| >>> from Bio import SeqIO |
| >>> with open("Quality/example.fastq") as handle: |
| ... for record in SeqIO.parse(handle, "fastq"): |
| ... print("%s %s" % (record.id, record.seq)) |
| EAS54_6_R1_2_1_413_324 CCCTTCTTGTCTTCAGCGTTTCTCC |
| EAS54_6_R1_2_1_540_792 TTGGCAGGCCAAGGCCGATGGATCA |
| EAS54_6_R1_2_1_443_348 GTTGCTTCTGGCGTGGGTGGGGGGG |
| |
| If you want to look at the qualities, they are record in each record's |
| per-letter-annotation dictionary as a simple list of integers: |
| |
| >>> print(record.letter_annotations["phred_quality"]) |
| [26, 26, 26, 26, 26, 26, 26, 26, 26, 26, 26, 24, 26, 22, 26, 26, 13, 22, 26, 18, 24, 18, 18, 18, 18] |
| |
| To modify the records returned by the parser, you can use a generator |
| function. For example, to store the mean PHRED quality in the record |
| description, use |
| |
| >>> from statistics import mean |
| >>> def modify_records(records): |
| ... for record in records: |
| ... record.description = mean(record.letter_annotations['phred_quality']) |
| ... yield record |
| ... |
| >>> with open('Quality/example.fastq') as handle: |
| ... for record in modify_records(FastqPhredIterator(handle)): |
| ... print(record.id, record.description) |
| ... |
| EAS54_6_R1_2_1_413_324 25.28 |
| EAS54_6_R1_2_1_540_792 24.52 |
| EAS54_6_R1_2_1_443_348 23.4 |
| |
| """ |
| if alphabet is not None: |
| raise ValueError("The alphabet argument is no longer supported") |
| super().__init__(source) |
|
|
|
|
| class FastqSolexaIterator(FastqIteratorAbstractBaseClass): |
| """Parser for old Solexa/Illumina FASTQ like files. |
| |
| These files differ in the quality mapping. |
| """ |
|
|
| |
| q_mapping = bytes( |
| ( |
| (letter - SOLEXA_SCORE_OFFSET) % 256 |
| if SOLEXA_SCORE_OFFSET - 5 <= letter < 63 + SOLEXA_SCORE_OFFSET |
| else INVALID_CHAR_CODE |
| ) |
| for letter in range(256) |
| ) |
|
|
| q_key = "solexa_quality" |
|
|
| def __init__( |
| self, |
| source: _TextIOSource, |
| alphabet: None = None, |
| ): |
| r"""Iterate over FASTQ records as SeqRecord objects. |
| |
| Arguments: |
| - source - input stream opened in text mode, or a path to a file |
| - alphabet - optional alphabet, no longer used. Leave as None. |
| |
| For each sequence in Solexa/Illumina FASTQ files there is a matching |
| string encoding the Solexa integer qualities using ASCII values with an |
| offset of 64. Solexa scores are scaled differently to PHRED scores, |
| and Biopython will NOT perform any automatic conversion when loading. |
| |
| NOTE - This file format is used by the OLD versions of the Solexa/ |
| Illumina pipeline. See also the FastqIlluminaIterator function for the |
| NEW version. |
| |
| For example, consider a file containing these five records:: |
| |
| @SLXA-B3_649_FC8437_R1_1_1_610_79 |
| GATGTGCAATACCTTTGTAGAGGAA |
| +SLXA-B3_649_FC8437_R1_1_1_610_79 |
| YYYYYYYYYYYYYYYYYYWYWYYSU |
| @SLXA-B3_649_FC8437_R1_1_1_397_389 |
| GGTTTGAGAAAGAGAAATGAGATAA |
| +SLXA-B3_649_FC8437_R1_1_1_397_389 |
| YYYYYYYYYWYYYYWWYYYWYWYWW |
| @SLXA-B3_649_FC8437_R1_1_1_850_123 |
| GAGGGTGTTGATCATGATGATGGCG |
| +SLXA-B3_649_FC8437_R1_1_1_850_123 |
| YYYYYYYYYYYYYWYYWYYSYYYSY |
| @SLXA-B3_649_FC8437_R1_1_1_362_549 |
| GGAAACAAAGTTTTTCTCAACATAG |
| +SLXA-B3_649_FC8437_R1_1_1_362_549 |
| YYYYYYYYYYYYYYYYYYWWWWYWY |
| @SLXA-B3_649_FC8437_R1_1_1_183_714 |
| GTATTATTTAATGGCATACACTCAA |
| +SLXA-B3_649_FC8437_R1_1_1_183_714 |
| YYYYYYYYYYWYYYYWYWWUWWWQQ |
| |
| Using this module directly you might run: |
| |
| >>> with open("Quality/solexa_example.fastq") as handle: |
| ... for record in FastqSolexaIterator(handle): |
| ... print("%s %s" % (record.id, record.seq)) |
| SLXA-B3_649_FC8437_R1_1_1_610_79 GATGTGCAATACCTTTGTAGAGGAA |
| SLXA-B3_649_FC8437_R1_1_1_397_389 GGTTTGAGAAAGAGAAATGAGATAA |
| SLXA-B3_649_FC8437_R1_1_1_850_123 GAGGGTGTTGATCATGATGATGGCG |
| SLXA-B3_649_FC8437_R1_1_1_362_549 GGAAACAAAGTTTTTCTCAACATAG |
| SLXA-B3_649_FC8437_R1_1_1_183_714 GTATTATTTAATGGCATACACTCAA |
| |
| Typically however, you would call this via Bio.SeqIO instead with |
| "fastq-solexa" as the format: |
| |
| >>> from Bio import SeqIO |
| >>> with open("Quality/solexa_example.fastq") as handle: |
| ... for record in SeqIO.parse(handle, "fastq-solexa"): |
| ... print("%s %s" % (record.id, record.seq)) |
| SLXA-B3_649_FC8437_R1_1_1_610_79 GATGTGCAATACCTTTGTAGAGGAA |
| SLXA-B3_649_FC8437_R1_1_1_397_389 GGTTTGAGAAAGAGAAATGAGATAA |
| SLXA-B3_649_FC8437_R1_1_1_850_123 GAGGGTGTTGATCATGATGATGGCG |
| SLXA-B3_649_FC8437_R1_1_1_362_549 GGAAACAAAGTTTTTCTCAACATAG |
| SLXA-B3_649_FC8437_R1_1_1_183_714 GTATTATTTAATGGCATACACTCAA |
| |
| The qualities are recorded in each record's per-letter-annotation |
| dictionary as a simple list of integers: |
| |
| >>> print(record.letter_annotations["solexa_quality"]) |
| [25, 25, 25, 25, 25, 25, 25, 25, 25, 25, 23, 25, 25, 25, 25, 23, 25, 23, 23, 21, 23, 23, 23, 17, 17] |
| |
| These scores aren't very good, but they are high enough that they map |
| almost exactly onto PHRED scores: |
| |
| >>> print("%0.2f" % phred_quality_from_solexa(25)) |
| 25.01 |
| |
| Let's look at faked example read which is even worse, where there are |
| more noticeable differences between the Solexa and PHRED scores:: |
| |
| @slxa_0001_1_0001_01 |
| ACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTNNNNNN |
| +slxa_0001_1_0001_01 |
| hgfedcba`_^]\[ZYXWVUTSRQPONMLKJIHGFEDCBA@?>=<; |
| |
| Again, you would typically use Bio.SeqIO to read this file in (rather |
| than calling the Bio.SeqIO.QualtityIO module directly). Most FASTQ |
| files will contain thousands of reads, so you would normally use |
| Bio.SeqIO.parse() as shown above. This example has only as one entry, |
| so instead we can use the Bio.SeqIO.read() function: |
| |
| >>> from Bio import SeqIO |
| >>> with open("Quality/solexa_faked.fastq") as handle: |
| ... record = SeqIO.read(handle, "fastq-solexa") |
| >>> print("%s %s" % (record.id, record.seq)) |
| slxa_0001_1_0001_01 ACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTNNNNNN |
| >>> print(record.letter_annotations["solexa_quality"]) |
| [40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0, -1, -2, -3, -4, -5] |
| |
| These quality scores are so low that when converted from the Solexa |
| scheme into PHRED scores they look quite different: |
| |
| >>> print("%0.2f" % phred_quality_from_solexa(-1)) |
| 2.54 |
| >>> print("%0.2f" % phred_quality_from_solexa(-5)) |
| 1.19 |
| |
| Note you can use the Bio.SeqIO.write() function or the SeqRecord's |
| format method to output the record(s): |
| |
| >>> print(record.format("fastq-solexa")) |
| @slxa_0001_1_0001_01 |
| ACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTNNNNNN |
| + |
| hgfedcba`_^]\[ZYXWVUTSRQPONMLKJIHGFEDCBA@?>=<; |
| <BLANKLINE> |
| |
| Note this output is slightly different from the input file as Biopython |
| has left out the optional repetition of the sequence identifier on the |
| "+" line. If you want the to use PHRED scores, use "fastq" or "qual" |
| as the output format instead, and Biopython will do the conversion for |
| you: |
| |
| >>> print(record.format("fastq")) |
| @slxa_0001_1_0001_01 |
| ACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTNNNNNN |
| + |
| IHGFEDCBA@?>=<;:9876543210/.-,++*)('&&%%$$##"" |
| <BLANKLINE> |
| |
| >>> print(record.format("qual")) |
| >slxa_0001_1_0001_01 |
| 40 39 38 37 36 35 34 33 32 31 30 29 28 27 26 25 24 23 22 21 |
| 20 19 18 17 16 15 14 13 12 11 10 10 9 8 7 6 5 5 4 4 3 3 2 2 |
| 1 1 |
| <BLANKLINE> |
| |
| As shown above, the poor quality Solexa reads have been mapped to the |
| equivalent PHRED score (e.g. -5 to 1 as shown earlier). |
| """ |
| if alphabet is not None: |
| raise ValueError("The alphabet argument is no longer supported") |
| super().__init__(source) |
|
|
|
|
| class FastqIlluminaIterator(FastqIteratorAbstractBaseClass): |
| """Parser for Illumina 1.3 to 1.7 FASTQ like files. |
| |
| These files differ in the quality mapping. |
| """ |
|
|
| q_mapping = bytes( |
| ( |
| letter - SOLEXA_SCORE_OFFSET |
| if SOLEXA_SCORE_OFFSET <= letter < 63 + SOLEXA_SCORE_OFFSET |
| else INVALID_CHAR_CODE |
| ) |
| for letter in range(256) |
| ) |
|
|
| q_key = "phred_quality" |
|
|
| def __init__( |
| self, |
| source: _TextIOSource, |
| alphabet: None = None, |
| ): |
| """Iterate over FASTQ records as SeqRecord objects. |
| |
| Arguments: |
| - source - input stream opened in text mode, or a path to a file |
| - alphabet - optional alphabet, no longer used. Leave as None. |
| |
| For each sequence in Illumina 1.3+ FASTQ files there is a matching |
| string encoding PHRED integer qualities using ASCII values with an |
| offset of 64. |
| |
| >>> from Bio import SeqIO |
| >>> record = SeqIO.read("Quality/illumina_faked.fastq", "fastq-illumina") |
| >>> print("%s %s" % (record.id, record.seq)) |
| Test ACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTN |
| >>> max(record.letter_annotations["phred_quality"]) |
| 40 |
| >>> min(record.letter_annotations["phred_quality"]) |
| 0 |
| |
| NOTE - Older versions of the Solexa/Illumina pipeline encoded Solexa |
| scores with an ASCII offset of 64. They are approximately equal but |
| only for high quality reads. If you have an old Solexa/Illumina file |
| with negative Solexa scores, and try and read this as an Illumina 1.3+ |
| file it will fail: |
| |
| >>> record2 = SeqIO.read("Quality/solexa_faked.fastq", "fastq-illumina") |
| Traceback (most recent call last): |
| ... |
| Bio.SeqIO.QualityIO.InvalidCharError: Invalid character (?) or (0x3f) in quality string not in correct range (are you sure you're using the right QualityIO parser?) with context: [...BA@?>=<...] |
| |
| NOTE - True Sanger style FASTQ files use PHRED scores with an offset |
| of 33. |
| """ |
| if alphabet is not None: |
| raise ValueError("The alphabet argument is no longer supported") |
| super().__init__(source) |
|
|
|
|
| class QualPhredIterator(SequenceIterator): |
| """Parser for QUAL files with PHRED quality scores but no sequence.""" |
|
|
| modes = "t" |
|
|
| def __init__( |
| self, |
| source: _TextIOSource, |
| alphabet: None = None, |
| ) -> None: |
| """For QUAL files which include PHRED quality scores, but no sequence. |
| |
| For example, consider this short QUAL file:: |
| |
| >EAS54_6_R1_2_1_413_324 |
| 26 26 18 26 26 26 26 26 26 26 26 26 26 26 26 22 26 26 26 26 |
| 26 26 26 23 23 |
| >EAS54_6_R1_2_1_540_792 |
| 26 26 26 26 26 26 26 26 26 26 26 22 26 26 26 26 26 12 26 26 |
| 26 18 26 23 18 |
| >EAS54_6_R1_2_1_443_348 |
| 26 26 26 26 26 26 26 26 26 26 26 24 26 22 26 26 13 22 26 18 |
| 24 18 18 18 18 |
| |
| Using this module directly you might run: |
| |
| >>> with open("Quality/example.qual") as handle: |
| ... for record in QualPhredIterator(handle): |
| ... print("%s read of length %d" % (record.id, len(record.seq))) |
| EAS54_6_R1_2_1_413_324 read of length 25 |
| EAS54_6_R1_2_1_540_792 read of length 25 |
| EAS54_6_R1_2_1_443_348 read of length 25 |
| |
| Typically however, you would call this via Bio.SeqIO instead with "qual" |
| as the format: |
| |
| >>> from Bio import SeqIO |
| >>> with open("Quality/example.qual") as handle: |
| ... for record in SeqIO.parse(handle, "qual"): |
| ... print("%s read of length %d" % (record.id, len(record.seq))) |
| EAS54_6_R1_2_1_413_324 read of length 25 |
| EAS54_6_R1_2_1_540_792 read of length 25 |
| EAS54_6_R1_2_1_443_348 read of length 25 |
| |
| Only the sequence length is known, as the QUAL file does not contain |
| the sequence string itself. |
| |
| The quality scores themselves are available as a list of integers |
| in each record's per-letter-annotation: |
| |
| >>> print(record.letter_annotations["phred_quality"]) |
| [26, 26, 26, 26, 26, 26, 26, 26, 26, 26, 26, 24, 26, 22, 26, 26, 13, 22, 26, 18, 24, 18, 18, 18, 18] |
| |
| You can still slice one of these SeqRecord objects: |
| |
| >>> sub_record = record[5:10] |
| >>> print("%s %s" % (sub_record.id, sub_record.letter_annotations["phred_quality"])) |
| EAS54_6_R1_2_1_443_348 [26, 26, 26, 26, 26] |
| |
| As of Biopython 1.59, this parser will accept files with negatives quality |
| scores but will replace them with the lowest possible PHRED score of zero. |
| This will trigger a warning, previously it raised a ValueError exception. |
| """ |
| if alphabet is not None: |
| raise ValueError("The alphabet argument is no longer supported") |
| super().__init__(source, fmt="QUAL") |
| |
| for line in self.stream: |
| if line[0] == ">": |
| break |
| else: |
| line = None |
| self._line = line |
|
|
| def __next__(self) -> SeqRecord: |
| """Parse the file and generate SeqRecord objects.""" |
|
|
| line = self._line |
| if line is None: |
| raise StopIteration |
| while True: |
| descr = line[1:].rstrip() |
| id = descr.split()[0] |
| name = id |
|
|
| qualities: list[int] = [] |
| for line in self.stream: |
| if line[0] == ">": |
| break |
| qualities.extend(int(word) for word in line.split()) |
| else: |
| line = None |
| self._line = line |
|
|
| if qualities and min(qualities) < 0: |
| warnings.warn( |
| "Negative quality score %i found, substituting PHRED zero instead." |
| % min(qualities), |
| BiopythonParserWarning, |
| ) |
| qualities = [max(0, q) for q in qualities] |
|
|
| |
| sequence = Seq(None, length=len(qualities)) |
|
|
| |
| record = SeqRecord._from_validated( |
| sequence, |
| id=id, |
| name=name, |
| description=descr, |
| letter_annotations={"phred_quality": qualities}, |
| ) |
| return record |
|
|
|
|
| assert SANGER_SCORE_OFFSET == ord("!") |
|
|
|
|
| class FastqPhredWriter(SequenceWriter): |
| """Class to write standard FASTQ format files (using PHRED quality scores). |
| |
| Although you can use this class directly, you are strongly encouraged |
| to use the top level ``Bio.SeqIO.write()`` function instead via the format |
| name "fastq" or the alias "fastq-sanger". |
| |
| For example, this code reads in a standard Sanger style FASTQ file |
| (using PHRED scores) and re-saves it as another Sanger style FASTQ file: |
| |
| >>> from Bio import SeqIO |
| >>> record_iterator = SeqIO.parse("Quality/example.fastq", "fastq") |
| >>> with open("Quality/temp.fastq", "w") as out_handle: |
| ... SeqIO.write(record_iterator, out_handle, "fastq") |
| 3 |
| |
| You might want to do this if the original file included extra line breaks, |
| which while valid may not be supported by all tools. The output file from |
| Biopython will have each sequence on a single line, and each quality |
| string on a single line (which is considered desirable for maximum |
| compatibility). |
| |
| In this next example, an old style Solexa/Illumina FASTQ file (using Solexa |
| quality scores) is converted into a standard Sanger style FASTQ file using |
| PHRED qualities: |
| |
| >>> from Bio import SeqIO |
| >>> record_iterator = SeqIO.parse("Quality/solexa_example.fastq", "fastq-solexa") |
| >>> with open("Quality/temp.fastq", "w") as out_handle: |
| ... SeqIO.write(record_iterator, out_handle, "fastq") |
| 5 |
| |
| This code is also called if you use the .format("fastq") method of a |
| SeqRecord, or .format("fastq-sanger") if you prefer that alias. |
| |
| Note that Sanger FASTQ files have an upper limit of PHRED quality 93, which is |
| encoded as ASCII 126, the tilde. If your quality scores are truncated to fit, a |
| warning is issued. |
| |
| P.S. To avoid cluttering up your working directory, you can delete this |
| temporary file now: |
| |
| >>> import os |
| >>> os.remove("Quality/temp.fastq") |
| """ |
|
|
| modes = "t" |
|
|
| @classmethod |
| def to_string(cls, record): |
| """Turn a SeqRecord into a Sanger FASTQ formatted string, and return it.""" |
| |
| seq_str = _get_seq_string(record) |
| qualities_str = _get_sanger_quality_str(record) |
| if len(qualities_str) != len(seq_str): |
| raise ValueError( |
| "Record %s has sequence length %i but %i quality scores" |
| % (record.id, len(seq_str), len(qualities_str)) |
| ) |
| id_ = _clean(record.id) if record.id else "" |
| description = _clean(record.description) |
| if description and description.split(None, 1)[0] == id_: |
| title = description |
| elif description: |
| title = f"{id_} {description}" |
| else: |
| title = id_ |
| return f"@{title}\n{seq_str}\n+\n{qualities_str}\n" |
|
|
| def write_record(self, record: SeqRecord) -> None: |
| """Write a single FASTQ record to the file.""" |
| self.handle.write(self.to_string(record)) |
|
|
|
|
| def as_fastq(record: SeqRecord) -> str: |
| """Turn a SeqRecord into a Sanger FASTQ formatted string, and return it.""" |
| warnings.warn( |
| """\ |
| QualityIO.as_fastq is deprecated. |
| |
| Instead of |
| |
| QualityIO.as_fastq(record) |
| |
| please use |
| |
| format(record, "fastq") |
| """, |
| DeprecationWarning, |
| ) |
| return FastqPhredWriter.to_string(record) |
|
|
|
|
| class QualPhredWriter(SequenceWriter): |
| """Class to write QUAL format files (using PHRED quality scores). |
| |
| Although you can use this class directly, you are strongly encouraged |
| to use the top level ``Bio.SeqIO.write()`` function instead. |
| |
| For example, this code reads in a FASTQ file and saves the quality scores |
| into a QUAL file: |
| |
| >>> from Bio import SeqIO |
| >>> record_iterator = SeqIO.parse("Quality/example.fastq", "fastq") |
| >>> with open("Quality/temp.qual", "w") as out_handle: |
| ... SeqIO.write(record_iterator, out_handle, "qual") |
| 3 |
| |
| This code is also called if you use the .format("qual") method of a |
| SeqRecord. |
| |
| P.S. Don't forget to clean up the temp file if you don't need it anymore: |
| |
| >>> import os |
| >>> os.remove("Quality/temp.qual") |
| """ |
|
|
| modes = "t" |
|
|
| def __init__( |
| self, |
| handle: _TextIOSource, |
| wrap: int = 60, |
| record2title: Callable[[SeqRecord], str] | None = None, |
| ) -> None: |
| """Create a QUAL writer. |
| |
| Arguments: |
| - handle - Handle to an output file, e.g. as returned |
| by open(filename, "w") |
| - wrap - Optional line length used to wrap sequence lines. |
| Defaults to wrapping the sequence at 60 characters. Use |
| zero (or None) for no wrapping, giving a single long line |
| for the sequence. |
| - record2title - Optional function to return the text to be |
| used for the title line of each record. By default a |
| combination of the record.id and record.description is |
| used. If the record.description starts with the record.id, |
| then just the record.description is used. |
| |
| The record2title argument is present for consistency with the |
| Bio.SeqIO.FastaIO writer class. |
| """ |
| super().__init__(handle) |
| |
| self.wrap: int | None = None |
| if wrap: |
| if wrap < 1: |
| raise ValueError |
| self.wrap = wrap |
| self.record2title = record2title |
|
|
| @classmethod |
| def to_string(cls, record: SeqRecord) -> str: |
| """Turn a SeqRecord into a QUAL formatted string.""" |
| id_ = _clean(record.id) if record.id else "" |
| description = _clean(record.description) |
| if description and description.split(None, 1)[0] == id_: |
| title = description |
| elif description: |
| title = f"{id_} {description}" |
| else: |
| title = id_ |
| lines = [f">{title}\n"] |
|
|
| qualities = _get_phred_quality(record) |
| try: |
| |
| |
| qualities_strs = [("%i" % round(q, 0)) for q in qualities] |
| except TypeError: |
| if None in qualities: |
| raise TypeError("A quality value of None was found") from None |
| else: |
| raise |
|
|
| |
| while qualities_strs: |
| line = qualities_strs.pop(0) |
| while qualities_strs and len(line) + 1 + len(qualities_strs[0]) < 60: |
| line += " " + qualities_strs.pop(0) |
| lines.append(line + "\n") |
| return "".join(lines) |
|
|
| def write_record(self, record: SeqRecord) -> None: |
| """Write a single QUAL record to the file.""" |
| handle = self.handle |
| wrap = self.wrap |
|
|
| if self.record2title: |
| title = self.clean(self.record2title(record)) |
| else: |
| id_ = self.clean(record.id) if record.id else "" |
| description = self.clean(record.description) |
| if description and description.split(None, 1)[0] == id_: |
| |
| title = description |
| elif description: |
| title = f"{id_} {description}" |
| else: |
| title = id_ |
| handle.write(f">{title}\n") |
|
|
| qualities = _get_phred_quality(record) |
| try: |
| |
| |
| qualities_strs = [("%i" % round(q, 0)) for q in qualities] |
| except TypeError: |
| if None in qualities: |
| raise TypeError("A quality value of None was found") from None |
| else: |
| raise |
|
|
| if wrap is not None and wrap > 5: |
| |
| data = " ".join(qualities_strs) |
| while True: |
| if len(data) <= wrap: |
| self.handle.write(data + "\n") |
| break |
| else: |
| |
| |
| i = data.rfind(" ", 0, wrap) |
| handle.write(data[:i] + "\n") |
| data = data[i + 1 :] |
| elif wrap: |
| |
| while qualities_strs: |
| line = qualities_strs.pop(0) |
| while qualities_strs and len(line) + 1 + len(qualities_strs[0]) < wrap: |
| line += " " + qualities_strs.pop(0) |
| handle.write(line + "\n") |
| else: |
| |
| data = " ".join(qualities_strs) |
| handle.write(data + "\n") |
|
|
|
|
| def as_qual(record: SeqRecord) -> str: |
| """Turn a SeqRecord into a QUAL formatted string.""" |
| warnings.warn( |
| """\ |
| QualityIO.as_qual is deprecated. |
| |
| Instead of |
| |
| QualityIO.as_qual(record) |
| |
| please use |
| |
| format(record, "qual") |
| """, |
| DeprecationWarning, |
| ) |
| return QualPhredWriter.to_string(record) |
|
|
|
|
| class FastqSolexaWriter(SequenceWriter): |
| r"""Write old style Solexa/Illumina FASTQ format files (with Solexa qualities). |
| |
| This outputs FASTQ files like those from the early Solexa/Illumina |
| pipeline, using Solexa scores and an ASCII offset of 64. These are |
| NOT compatible with the standard Sanger style PHRED FASTQ files. |
| |
| If your records contain a "solexa_quality" entry under letter_annotations, |
| this is used, otherwise any "phred_quality" entry will be used after |
| conversion using the solexa_quality_from_phred function. If neither style |
| of quality scores are present, an exception is raised. |
| |
| Although you can use this class directly, you are strongly encouraged |
| to use the ``as_fastq_solexa`` function, or top-level ``Bio.SeqIO.write()`` |
| function instead. For example, this code reads in a FASTQ file and re-saves |
| it as another FASTQ file: |
| |
| >>> from Bio import SeqIO |
| >>> record_iterator = SeqIO.parse("Quality/solexa_example.fastq", "fastq-solexa") |
| >>> with open("Quality/temp.fastq", "w") as out_handle: |
| ... SeqIO.write(record_iterator, out_handle, "fastq-solexa") |
| 5 |
| |
| You might want to do this if the original file included extra line breaks, |
| which (while valid) may not be supported by all tools. The output file |
| from Biopython will have each sequence on a single line, and each quality |
| string on a single line (which is considered desirable for maximum |
| compatibility). |
| |
| This code is also called if you use the .format("fastq-solexa") method of |
| a SeqRecord. For example, |
| |
| >>> record = SeqIO.read("Quality/sanger_faked.fastq", "fastq-sanger") |
| >>> print(record.format("fastq-solexa")) |
| @Test PHRED qualities from 40 to 0 inclusive |
| ACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTN |
| + |
| hgfedcba`_^]\[ZYXWVUTSRQPONMLKJHGFECB@>;; |
| <BLANKLINE> |
| |
| Note that Solexa FASTQ files have an upper limit of Solexa quality 62, which is |
| encoded as ASCII 126, the tilde. If your quality scores must be truncated to fit, |
| a warning is issued. |
| |
| P.S. Don't forget to delete the temp file if you don't need it anymore: |
| |
| >>> import os |
| >>> os.remove("Quality/temp.fastq") |
| """ |
|
|
| modes = "t" |
|
|
| @classmethod |
| def to_string(cls, record: SeqRecord) -> str: |
| """Turn a SeqRecord into a Solexa FASTQ formatted string. |
| |
| This is used internally by the SeqRecord's .format("fastq-solexa") |
| method and by the SeqIO.write(..., ..., "fastq-solexa") function. |
| """ |
| |
| seq_str = _get_seq_string(record) |
| qualities_str = _get_solexa_quality_str(record) |
| if len(qualities_str) != len(seq_str): |
| raise ValueError( |
| "Record %s has sequence length %i but %i quality scores" |
| % (record.id, len(seq_str), len(qualities_str)) |
| ) |
| id_ = _clean(record.id) if record.id else "" |
| description = _clean(record.description) |
| if description and description.split(None, 1)[0] == id_: |
| |
| title = description |
| elif description: |
| title = f"{id_} {description}" |
| else: |
| title = id_ |
| return f"@{title}\n{seq_str}\n+\n{qualities_str}\n" |
|
|
| def write_record(self, record: SeqRecord) -> None: |
| """Write a single FASTQ record to the file.""" |
| self.handle.write(self.to_string(record)) |
|
|
|
|
| def as_fastq_solexa(record: SeqRecord) -> str: |
| """Turn a SeqRecord into a Solexa FASTQ formatted string.""" |
| warnings.warn( |
| """\ |
| QualityIO.as_fastq_solexa is deprecated. |
| |
| Instead of |
| |
| QualityIO.as_fastq_solexa(record) |
| |
| please use |
| |
| format(record, "fastq-solexa") |
| """, |
| DeprecationWarning, |
| ) |
| return FastqSolexaWriter.to_string(record) |
|
|
|
|
| class FastqIlluminaWriter(SequenceWriter): |
| r"""Write Illumina 1.3+ FASTQ format files (with PHRED quality scores). |
| |
| This outputs FASTQ files like those from the Solexa/Illumina 1.3+ pipeline, |
| using PHRED scores and an ASCII offset of 64. Note these files are NOT |
| compatible with the standard Sanger style PHRED FASTQ files which use an |
| ASCII offset of 32. |
| |
| Although you can use this class directly, you are strongly encouraged to |
| use the ``as_fastq_illumina`` or top-level ``Bio.SeqIO.write()`` function |
| with format name "fastq-illumina" instead. This code is also called if you |
| use the .format("fastq-illumina") method of a SeqRecord. For example, |
| |
| >>> from Bio import SeqIO |
| >>> record = SeqIO.read("Quality/sanger_faked.fastq", "fastq-sanger") |
| >>> print(record.format("fastq-illumina")) |
| @Test PHRED qualities from 40 to 0 inclusive |
| ACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTN |
| + |
| hgfedcba`_^]\[ZYXWVUTSRQPONMLKJIHGFEDCBA@ |
| <BLANKLINE> |
| |
| Note that Illumina FASTQ files have an upper limit of PHRED quality 62, which is |
| encoded as ASCII 126, the tilde. If your quality scores are truncated to fit, a |
| warning is issued. |
| """ |
|
|
| modes = "t" |
|
|
| @classmethod |
| def to_string(cls, record: SeqRecord) -> str: |
| """Turn a SeqRecord into an Illumina FASTQ formatted string. |
| |
| This is used internally by the SeqRecord's .format("fastq-illumina") |
| method and by the SeqIO.write(..., ..., "fastq-illumina") function. |
| """ |
| |
| seq_str = _get_seq_string(record) |
| qualities_str = _get_illumina_quality_str(record) |
| if len(qualities_str) != len(seq_str): |
| raise ValueError( |
| "Record %s has sequence length %i but %i quality scores" |
| % (record.id, len(seq_str), len(qualities_str)) |
| ) |
| id_ = _clean(record.id) if record.id else "" |
| description = _clean(record.description) |
| if description and description.split(None, 1)[0] == id_: |
| title = description |
| elif description: |
| title = f"{id_} {description}" |
| else: |
| title = id_ |
| return f"@{title}\n{seq_str}\n+\n{qualities_str}\n" |
|
|
| def write_record(self, record: SeqRecord) -> None: |
| """Write a single FASTQ record to the file.""" |
| self.handle.write(self.to_string(record)) |
|
|
|
|
| def as_fastq_illumina(record: SeqRecord) -> str: |
| """Turn a SeqRecord into an Illumina FASTQ formatted string.""" |
| warnings.warn( |
| """\ |
| QualityIO.as_fastq_illumina is deprecated. |
| |
| Instead of |
| |
| QualityIO.as_fastq_illumina(record) |
| |
| please use |
| |
| format(record, "fastq-illumina") |
| """, |
| DeprecationWarning, |
| ) |
| return FastqIlluminaWriter.to_string(record) |
|
|
|
|
| def PairedFastaQualIterator( |
| fasta_source: _TextIOSource, |
| qual_source: _TextIOSource, |
| alphabet: None = None, |
| ) -> Iterator[SeqRecord]: |
| """Iterate over matched FASTA and QUAL files as SeqRecord objects. |
| |
| For example, consider this short QUAL file with PHRED quality scores:: |
| |
| >EAS54_6_R1_2_1_413_324 |
| 26 26 18 26 26 26 26 26 26 26 26 26 26 26 26 22 26 26 26 26 |
| 26 26 26 23 23 |
| >EAS54_6_R1_2_1_540_792 |
| 26 26 26 26 26 26 26 26 26 26 26 22 26 26 26 26 26 12 26 26 |
| 26 18 26 23 18 |
| >EAS54_6_R1_2_1_443_348 |
| 26 26 26 26 26 26 26 26 26 26 26 24 26 22 26 26 13 22 26 18 |
| 24 18 18 18 18 |
| |
| And a matching FASTA file:: |
| |
| >EAS54_6_R1_2_1_413_324 |
| CCCTTCTTGTCTTCAGCGTTTCTCC |
| >EAS54_6_R1_2_1_540_792 |
| TTGGCAGGCCAAGGCCGATGGATCA |
| >EAS54_6_R1_2_1_443_348 |
| GTTGCTTCTGGCGTGGGTGGGGGGG |
| |
| You can parse these separately using Bio.SeqIO with the "qual" and |
| "fasta" formats, but then you'll get a group of SeqRecord objects with |
| no sequence, and a matching group with the sequence but not the |
| qualities. Because it only deals with one input file handle, Bio.SeqIO |
| can't be used to read the two files together - but this function can! |
| For example, |
| |
| >>> with open("Quality/example.fasta") as f: |
| ... with open("Quality/example.qual") as q: |
| ... for record in PairedFastaQualIterator(f, q): |
| ... print("%s %s" % (record.id, record.seq)) |
| ... |
| EAS54_6_R1_2_1_413_324 CCCTTCTTGTCTTCAGCGTTTCTCC |
| EAS54_6_R1_2_1_540_792 TTGGCAGGCCAAGGCCGATGGATCA |
| EAS54_6_R1_2_1_443_348 GTTGCTTCTGGCGTGGGTGGGGGGG |
| |
| As with the FASTQ or QUAL parsers, if you want to look at the qualities, |
| they are in each record's per-letter-annotation dictionary as a simple |
| list of integers: |
| |
| >>> print(record.letter_annotations["phred_quality"]) |
| [26, 26, 26, 26, 26, 26, 26, 26, 26, 26, 26, 24, 26, 22, 26, 26, 13, 22, 26, 18, 24, 18, 18, 18, 18] |
| |
| If you have access to data as a FASTQ format file, using that directly |
| would be simpler and more straight forward. Note that you can easily use |
| this function to convert paired FASTA and QUAL files into FASTQ files: |
| |
| >>> from Bio import SeqIO |
| >>> with open("Quality/example.fasta") as f: |
| ... with open("Quality/example.qual") as q: |
| ... SeqIO.write(PairedFastaQualIterator(f, q), "Quality/temp.fastq", "fastq") |
| ... |
| 3 |
| |
| And don't forget to clean up the temp file if you don't need it anymore: |
| |
| >>> import os |
| >>> os.remove("Quality/temp.fastq") |
| """ |
| if alphabet is not None: |
| raise ValueError("The alphabet argument is no longer supported") |
|
|
| from Bio.SeqIO.FastaIO import FastaIterator |
|
|
| fasta_iter = FastaIterator(fasta_source) |
| qual_iter = QualPhredIterator(qual_source) |
|
|
| |
| |
| while True: |
| try: |
| f_rec = next(fasta_iter) |
| except StopIteration: |
| f_rec = None |
| try: |
| q_rec = next(qual_iter) |
| except StopIteration: |
| q_rec = None |
| if f_rec is None and q_rec is None: |
| |
| break |
| if f_rec is None: |
| raise ValueError("FASTA file has more entries than the QUAL file.") |
| if q_rec is None: |
| raise ValueError("QUAL file has more entries than the FASTA file.") |
| if f_rec.id != q_rec.id: |
| raise ValueError( |
| f"FASTA and QUAL entries do not match ({f_rec.id} vs {q_rec.id})." |
| ) |
| if len(f_rec) != len(q_rec.letter_annotations["phred_quality"]): |
| raise ValueError( |
| f"Sequence length and number of quality scores disagree for {f_rec.id}" |
| ) |
| |
| f_rec.letter_annotations["phred_quality"] = q_rec.letter_annotations[ |
| "phred_quality" |
| ] |
| yield f_rec |
| |
|
|
|
|
| def _fastq_generic( |
| in_file: _TextIOSource, |
| out_file: _TextIOSource, |
| mapping: Sequence[str] | Mapping[int, str | int | None], |
| ) -> int: |
| """FASTQ helper function where can't have data loss by truncation (PRIVATE).""" |
| |
| count = 0 |
| null = chr(0) |
| with as_handle(out_file, "w") as out_handle: |
| for title, seq, old_qual in FastqGeneralIterator(in_file): |
| count += 1 |
| |
| qual = old_qual.translate(mapping) |
| if null in qual: |
| invalid_index = qual.find(null) |
| raise InvalidCharError( |
| old_qual, |
| invalid_index, |
| details="not in correct range (are you sure you're using the right QualityIO parser?)", |
| ) |
|
|
| out_handle.write(f"@{title}\n{seq}\n+\n{qual}\n") |
| return count |
|
|
|
|
| def _fastq_generic2( |
| in_file: _TextIOSource, |
| out_file: _TextIOSource, |
| mapping: Sequence[str] | Mapping[int, str | int | None], |
| truncate_char: str, |
| truncate_msg: str, |
| ) -> int: |
| """FASTQ helper function where there could be data loss by truncation (PRIVATE).""" |
| |
| count = 0 |
| null = chr(0) |
| with as_handle(out_file, "w") as out_handle: |
| for title, seq, old_qual in FastqGeneralIterator(in_file): |
| count += 1 |
| |
| qual = old_qual.translate(mapping) |
| if null in qual: |
| invalid_index = qual.find(null) |
| raise InvalidCharError( |
| old_qual, |
| invalid_index, |
| details="not in correct range (are you sure you're using the right QualityIO parser?)", |
| ) |
| if truncate_char in qual: |
| qual = qual.replace(truncate_char, chr(126)) |
| warnings.warn(truncate_msg, BiopythonWarning) |
| out_handle.write(f"@{title}\n{seq}\n+\n{qual}\n") |
| return count |
|
|
|
|
| def _fastq_sanger_convert_fastq_sanger( |
| in_file: _TextIOSource, out_file: _TextIOSource |
| ) -> int: |
| """Fast Sanger FASTQ to Sanger FASTQ conversion (PRIVATE). |
| |
| Useful for removing line wrapping and the redundant second identifier |
| on the plus lines. Will check also check the quality string is valid. |
| |
| Avoids creating SeqRecord and Seq objects in order to speed up this |
| conversion. |
| """ |
| |
| mapping = "".join( |
| [chr(0) for ascii in range(33)] |
| + [chr(ascii) for ascii in range(33, 127)] |
| + [chr(0) for ascii in range(127, 256)] |
| ) |
| assert len(mapping) == 256 |
| return _fastq_generic(in_file, out_file, mapping) |
|
|
|
|
| def _fastq_solexa_convert_fastq_solexa( |
| in_file: _TextIOSource, out_file: _TextIOSource |
| ) -> int: |
| """Fast Solexa FASTQ to Solexa FASTQ conversion (PRIVATE). |
| |
| Useful for removing line wrapping and the redundant second identifier |
| on the plus lines. Will check also check the quality string is valid. |
| Avoids creating SeqRecord and Seq objects in order to speed up this |
| conversion. |
| """ |
| |
| mapping = "".join( |
| [chr(0) for ascii in range(59)] |
| + [chr(ascii) for ascii in range(59, 127)] |
| + [chr(0) for ascii in range(127, 256)] |
| ) |
| assert len(mapping) == 256 |
| return _fastq_generic(in_file, out_file, mapping) |
|
|
|
|
| def _fastq_illumina_convert_fastq_illumina( |
| in_file: _TextIOSource, out_file: _TextIOSource |
| ) -> int: |
| """Fast Illumina 1.3+ FASTQ to Illumina 1.3+ FASTQ conversion (PRIVATE). |
| |
| Useful for removing line wrapping and the redundant second identifier |
| on the plus lines. Will check also check the quality string is valid. |
| Avoids creating SeqRecord and Seq objects in order to speed up this |
| conversion. |
| """ |
| |
| mapping = "".join( |
| [chr(0) for ascii in range(64)] |
| + [chr(ascii) for ascii in range(64, 127)] |
| + [chr(0) for ascii in range(127, 256)] |
| ) |
| assert len(mapping) == 256 |
| return _fastq_generic(in_file, out_file, mapping) |
|
|
|
|
| def _fastq_illumina_convert_fastq_sanger( |
| in_file: _TextIOSource, out_file: _TextIOSource |
| ) -> int: |
| """Fast Illumina 1.3+ FASTQ to Sanger FASTQ conversion (PRIVATE). |
| |
| Avoids creating SeqRecord and Seq objects in order to speed up this |
| conversion. |
| """ |
| |
| mapping = "".join( |
| [chr(0) for ascii in range(64)] |
| + [chr(33 + q) for q in range(62 + 1)] |
| + [chr(0) for ascii in range(127, 256)] |
| ) |
| assert len(mapping) == 256 |
| return _fastq_generic(in_file, out_file, mapping) |
|
|
|
|
| def _fastq_sanger_convert_fastq_illumina( |
| in_file: _TextIOSource, out_file: _TextIOSource |
| ) -> int: |
| """Fast Sanger FASTQ to Illumina 1.3+ FASTQ conversion (PRIVATE). |
| |
| Avoids creating SeqRecord and Seq objects in order to speed up this |
| conversion. Will issue a warning if the scores had to be truncated at 62 |
| (maximum possible in the Illumina 1.3+ FASTQ format) |
| """ |
| |
| trunc_char = chr(1) |
| mapping = "".join( |
| [chr(0) for ascii in range(33)] |
| + [chr(64 + q) for q in range(62 + 1)] |
| + [trunc_char for ascii in range(96, 127)] |
| + [chr(0) for ascii in range(127, 256)] |
| ) |
| assert len(mapping) == 256 |
| return _fastq_generic2( |
| in_file, |
| out_file, |
| mapping, |
| trunc_char, |
| "Data loss - max PHRED quality 62 in Illumina 1.3+ FASTQ", |
| ) |
|
|
|
|
| def _fastq_solexa_convert_fastq_sanger( |
| in_file: _TextIOSource, out_file: _TextIOSource |
| ) -> int: |
| """Fast Solexa FASTQ to Sanger FASTQ conversion (PRIVATE). |
| |
| Avoids creating SeqRecord and Seq objects in order to speed up this |
| conversion. |
| """ |
| |
| mapping = "".join( |
| [chr(0) for ascii in range(59)] |
| + [ |
| chr(33 + int(round(phred_quality_from_solexa(q)))) |
| for q in range(-5, 62 + 1) |
| ] |
| + [chr(0) for ascii in range(127, 256)] |
| ) |
| assert len(mapping) == 256 |
| return _fastq_generic(in_file, out_file, mapping) |
|
|
|
|
| def _fastq_sanger_convert_fastq_solexa( |
| in_file: _TextIOSource, out_file: _TextIOSource |
| ) -> int: |
| """Fast Sanger FASTQ to Solexa FASTQ conversion (PRIVATE). |
| |
| Avoids creating SeqRecord and Seq objects in order to speed up this |
| conversion. Will issue a warning if the scores had to be truncated at 62 |
| (maximum possible in the Solexa FASTQ format) |
| """ |
| |
| trunc_char = chr(1) |
| mapping = "".join( |
| [chr(0) for ascii in range(33)] |
| + [chr(64 + int(round(solexa_quality_from_phred(q)))) for q in range(62 + 1)] |
| + [trunc_char for ascii in range(96, 127)] |
| + [chr(0) for ascii in range(127, 256)] |
| ) |
| assert len(mapping) == 256 |
| return _fastq_generic2( |
| in_file, |
| out_file, |
| mapping, |
| trunc_char, |
| "Data loss - max Solexa quality 62 in Solexa FASTQ", |
| ) |
|
|
|
|
| def _fastq_solexa_convert_fastq_illumina( |
| in_file: _TextIOSource, out_file: _TextIOSource |
| ) -> int: |
| """Fast Solexa FASTQ to Illumina 1.3+ FASTQ conversion (PRIVATE). |
| |
| Avoids creating SeqRecord and Seq objects in order to speed up this |
| conversion. |
| """ |
| |
| mapping = "".join( |
| [chr(0) for ascii in range(59)] |
| + [ |
| chr(64 + int(round(phred_quality_from_solexa(q)))) |
| for q in range(-5, 62 + 1) |
| ] |
| + [chr(0) for ascii in range(127, 256)] |
| ) |
| assert len(mapping) == 256 |
| return _fastq_generic(in_file, out_file, mapping) |
|
|
|
|
| def _fastq_illumina_convert_fastq_solexa( |
| in_file: _TextIOSource, out_file: _TextIOSource |
| ) -> int: |
| """Fast Illumina 1.3+ FASTQ to Solexa FASTQ conversion (PRIVATE). |
| |
| Avoids creating SeqRecord and Seq objects in order to speed up this |
| conversion. |
| """ |
| |
| mapping = "".join( |
| [chr(0) for ascii in range(64)] |
| + [chr(64 + int(round(solexa_quality_from_phred(q)))) for q in range(62 + 1)] |
| + [chr(0) for ascii in range(127, 256)] |
| ) |
| assert len(mapping) == 256 |
| return _fastq_generic(in_file, out_file, mapping) |
|
|
|
|
| def _fastq_convert_fasta(in_file: _TextIOSource, out_file: _TextIOSource) -> int: |
| """Fast FASTQ to FASTA conversion (PRIVATE). |
| |
| Avoids dealing with the FASTQ quality encoding, and creating SeqRecord and |
| Seq objects in order to speed up this conversion. |
| |
| NOTE - This does NOT check the characters used in the FASTQ quality string |
| are valid! |
| """ |
| |
| count = 0 |
| with as_handle(out_file, "w") as out_handle: |
| for title, seq, qual in FastqGeneralIterator(in_file): |
| count += 1 |
| out_handle.write(f">{title}\n") |
| |
| for i in range(0, len(seq), 60): |
| out_handle.write(seq[i : i + 60] + "\n") |
| return count |
|
|
|
|
| def _fastq_convert_tab(in_file: _TextIOSource, out_file: _TextIOSource) -> int: |
| """Fast FASTQ to simple tabbed conversion (PRIVATE). |
| |
| Avoids dealing with the FASTQ quality encoding, and creating SeqRecord and |
| Seq objects in order to speed up this conversion. |
| |
| NOTE - This does NOT check the characters used in the FASTQ quality string |
| are valid! |
| """ |
| |
| count = 0 |
| with as_handle(out_file, "w") as out_handle: |
| for title, seq, qual in FastqGeneralIterator(in_file): |
| count += 1 |
| out_handle.write(f"{title.split(None, 1)[0]}\t{seq}\n") |
| return count |
|
|
|
|
| def _fastq_convert_qual( |
| in_file: _TextIOSource, |
| out_file: _TextIOSource, |
| mapping: Mapping[str, str], |
| ) -> int: |
| """FASTQ helper function for QUAL output (PRIVATE). |
| |
| Mapping should be a dictionary mapping expected ASCII characters from the |
| FASTQ quality string to PHRED quality scores (as strings). |
| """ |
| |
| count = 0 |
| with as_handle(out_file, "w") as out_handle: |
| for title, seq, qual in FastqGeneralIterator(in_file): |
| count += 1 |
| out_handle.write(f">{title}\n") |
| |
| try: |
| qualities_strs = [mapping[ascii_] for ascii_ in qual] |
| except KeyError: |
| invalid_index = _find_index_where(qual, lambda x: x not in mapping) |
| assert invalid_index >= 0, "Invalid char not in mapping not found!" |
| raise InvalidCharError( |
| qual, |
| invalid_index, |
| details="not in correct range (are you sure you're using the right QualityIO parser?)", |
| ) from None |
| data = " ".join(qualities_strs) |
| while len(data) > 60: |
| |
| |
| if data[60] == " ": |
| out_handle.write(data[:60] + "\n") |
| data = data[61:] |
| elif data[59] == " ": |
| out_handle.write(data[:59] + "\n") |
| data = data[60:] |
| else: |
| assert data[58] == " ", "Internal logic failure in wrapping" |
| out_handle.write(data[:58] + "\n") |
| data = data[59:] |
| out_handle.write(data + "\n") |
| return count |
|
|
|
|
| def _fastq_sanger_convert_qual(in_file: _TextIOSource, out_file: _TextIOSource) -> int: |
| """Fast Sanger FASTQ to QUAL conversion (PRIVATE).""" |
| mapping = {chr(q + 33): str(q) for q in range(93 + 1)} |
| return _fastq_convert_qual(in_file, out_file, mapping) |
|
|
|
|
| def _fastq_solexa_convert_qual(in_file: _TextIOSource, out_file: _TextIOSource) -> int: |
| """Fast Solexa FASTQ to QUAL conversion (PRIVATE).""" |
| mapping = { |
| chr(q + 64): str(int(round(phred_quality_from_solexa(q)))) |
| for q in range(-5, 62 + 1) |
| } |
| return _fastq_convert_qual(in_file, out_file, mapping) |
|
|
|
|
| def _fastq_illumina_convert_qual( |
| in_file: _TextIOSource, out_file: _TextIOSource |
| ) -> int: |
| """Fast Illumina 1.3+ FASTQ to QUAL conversion (PRIVATE).""" |
| mapping = {chr(q + 64): str(q) for q in range(62 + 1)} |
| return _fastq_convert_qual(in_file, out_file, mapping) |
|
|
|
|
| @dataclass |
| class InvalidCharError(ValueError): |
| """ |
| Custom error for strings that have a character that is invalid for whatever reason (eg: non-ascii, invalid range) |
| |
| Main attributes: |
| - full_string - the string which contains the invalid character (str) |
| - index - position of the invalid character in full_string (int) |
| - details - additional information to add to the error message. Like: 'not in correct range' (str) |
| - r - how many characters on each side of the invalid character to include in the error message (int) |
| """ |
|
|
| full_string: str |
| index: int |
| details: str |
| r: int = 3 |
|
|
| def __str__(self) -> str: |
| char = self.full_string[self.index] |
|
|
| surrounding_characters = self.full_string[ |
| max(self.index - self.r, 0) : self.index + self.r + 1 |
| ] |
| left_complete = self.index - self.r < 0 |
| prefix = "" if left_complete else "..." |
| right_complete = self.index + self.r + 1 >= len(self.full_string) |
| suffix = "" if right_complete else "..." |
|
|
| return f"Invalid character ({char}) or (0x{char.encode().hex()}) in quality string {self.details} with context: [{prefix}{surrounding_characters}{suffix}]" |
|
|
|
|
| def _find_index_where(iterable: Iterable, predicate: Callable[[Any], bool]) -> int: |
| for i, x in enumerate(iterable): |
| if predicate(x) is True: |
| return i |
|
|
| return -1 |
|
|
|
|
| if __name__ == "__main__": |
| from Bio._utils import run_doctest |
|
|
| run_doctest(verbose=0) |
|
|