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Data Analysis and Visualization |
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## Sequencing Libraries |
## Chromium Single Cell Gene Expression Libraries |
Chromium Single Cell 3' Gene Expression Dual Index libraries comprise standard Illumina paired-end constructs which begin with P5 and end with P7. These libraries include 16 bp 10x Barcodes at the start of TruSeq Read 1 while i7 and i5 sample index sequences are incorporated as the sample index read. TruSeq Read 1 an... |
## Chromium Single Cell 3' Gene Expression Library |
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Sequencing these libraries produces a standard Illumina BCL data output folder. |
## Illumina Sequencer Compatibility |
The compatibility of the listed sequencers has been verified by 10x Genomics. Some variation in assay performance is expected based on sequencer choice. For more information about performance variation, visit the 10x Genomics Support website. |
- l MiSeq |
- l NextSeq 500/550/2000 |
- l HiSeq 2500 (Rapid Run) |
- l HiSeq 3000/4000 |
- l NovaSeq 6000 |
## Sample Indices |
Each sample index in the relevant Dual Index Kit contains a mix of one unique i7 and one unique i5 sample index. If multiple samples are pooled in a sequence lane, the sample index name (i.e. the Dual Index plate well ID) is needed in the sample sheet used for generating FASTQs with "cellranger Tab 1 Text Tab 2 Text Ta... |
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## Library Sequencing Depth & Run Parameters |
## 3' Gene Expression Library |
| Parameter | Description | |
|------------------|------------------------------------| |
| Sequencing Depth | Minimum 20,000 read pairs per cell | |
| Sequencing Type | Paired-end, dual indexing | |
| Sequencing Read | Recommended Number of Cycles | |
| Read 1 | 28 cycles | |
| i7 Index | 10 cycles | |
| i5 Index | 10 cycles | |
| Read 2 | 90 cycles | |
## Library Loading |
Once quantified and normalized, libraries should be denatured and diluted as recommended for Illumina sequencing platforms. Refer to Illumina documentation for denaturing and diluting libraries. Refer to the 10x Genomics Support website for more information. |
## Library Loading |
| Instrument | Loading Concentration (pM)* | PhiX (%) | |
|-------------------------------------|-------------------------------|------------| |
| MiSeq | 12 | 1 | |
| NextSeq 500/550 | 1.6 | 1 | |
| NextSeq 1000/2000 | 650 | 1 | |
| HiSeq 2500 (RR) | 12 | 1 | |
| HiSeq 4000 | 240 | 1 | |
| NovaSeq 6000 Standard & Xp workflow | 150 | 1 | |
## Library Pooling |
Different libraries maybe pooled for sequencing, taking into account the differences in cell number and per-cell read depth requirements between each library. Samples utilizing the same sample index should not be pooled together Tab 1 Text Tab 2 Text Tab 3 Text Tab 4 Text Tab 5 Text Tab 6 Text or run on the same flow... |
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## Data Analysis and Visualization |
Sequencing data may be analyzed using Cell Ranger and visualized using Loupe Browser. Key features for these tools are listed below. For detailed product-specific information, visit the 10x Genomics Support website. |
## Cell Ranger |
Cell Ranger is a set of analysis pipelines that processes Chromium Single Gene Expression data to align reads, generate Feature Barcode matrices and perform clustering and gene expression analysis. |
- l Input: Binary base call (BCL) and FASTQ |
- l Output: BAM, MEX, CSV, HDF5, Web Summary, .cloupe/.loupe |
- l Operating System: Linux |
## Cloud Analysis |
Cloud Analysis is currently only available for US & Canada customers. |
Cloud Analysis allows users to run Cell Ranger analysis pipelines from a web browser while computation is handled in the cloud. |
- l Key features: scalable, highly secure, simple to set up and run |
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