Shulou(Shulou.com)06/02 Report--

How to use cell ranger to split 10X single-cell transcriptome raw data, I believe that many inexperienced people do not know what to do, so this paper summarizes the causes of the problem and solutions, through this article I hope you can solve this problem.

Cell ranger is a pipeline provided by 10x genomics company, which is specially used to analyze 10X single-cell transcriptome data. It includes many functions such as original data splitting, expression quantification, cluster analysis and so on. This paper mainly introduces how to use this software to split the original data.

Download the latest version of the software directly from the official website at the following URL

Https://support.10xgenomics.com/single-cell-gene-expression/software/downloads/latest

The software consists of several subcommands and splits the data through the mkfastq command. The flow chart is as follows

There are two ways to use it

Cellranger mkfastq\-id test\-run run_directory\-csv simple.csvcellranger mkfastq\-id test\-run run_directory\-samplesheet samplesheet.csv

The id parameter specifies the name of the output directory, and the run parameter specifies the directory where the original bcl file is located. This command is actually an encapsulation of the bcl2fastq command for splitting data provided by illumina. It requires sample name, index and other information, and supports two formats. One is the regular samplesheet.csv file of illlumina, which is in the following format.

The other is a simplified version of csv format customized by 10x genomics, which contains the following

Lane,Sample,Index1,test_sample,SI-GA-A3

There are only three columns, the first column specifies lane ID, the second column specifies the name of the sample, the third column specifies the name of index, and each index of 10X genomics represents four specific oligo sequences, as shown below

When determining a sample based on index, a mismatch of 1 to 2 bases is allowed. When actually splitting the data, it is more recommended to use a three-column CSV file, because the samplesheet file needs to modify the corresponding Reads information according to different versions of the kit.

The structure of the library generated by the V2 kit is as follows

The library structure generated by the V3 kit is as follows

Compared with V2, the length of UMI and PolyT used in V3 kit changed, which led to the discrepancy of sequence length between R1 and R2. The length of R1 end of V2 kit was 26bp, the length of R1 terminal of V2 kit was 26bp, the length of R1 terminal of V3 kit was 28bp, the length of R1 terminal of V3 kit was 28bp, the length of R1 terminal of V3 kit was 98bp, and the R2 end of V3 kit was 91bp.

If you use a samplesheet file, you need to adjust the sequence length in [Reads], while with a simplified version of the csv file, cell ranger can identify the version of the kit used and then automatically adjust the reads length.

The directory structure after the split is as follows

├── fastq_path │ ├── H35KCBCXY │ │ └── test_sample │ │ ├── test_sample_S1_L001_I1_001.fastq.gz │ │ ├── test_sample_S1_L001_R1_001.fastq.gz │ │ └── test_sample_S1_L001_R2_001.fastq.gz

For each sample, in addition to the common R1 and R2 terminal sequences, there is also an I1 sequence file, in which the index sequence is saved, as shown below

@ D00547:905:H35KCBCXY:1:1101:19188:87078 1 VOGATCGGGAGATCGGGGG.

Welcome to subscribe "Shulou Technology Information " to get latest news, interesting things and hot topics in the IT industry, and controls the hottest and latest Internet news, technology news and IT industry trends.