Single-cell sequencing data analysis (3) - download and use of cell ranger

Reference:
Single-cell combat (three) Cell Ranger use of the first exploration

The next step is to use the cell rangerhardwareTo analyze, but there is a prerequisite, we need to pay attention to, the system performance to be very good, generally less need for us to do, a lot of articles say here just need to understand, know the general process on the line.

System Requirements:
Linux At least 8 cores
CPU (16 cores recommended) 64GB
RAM (128GB recommended), minimum 64G, allows cellranger aggr to merge up to 250k cells
1T hard disk
64-bit CentOS/RedHat 6.0 or Ubuntu 12.04

The cell ranger software is divided into four main sections:
cellranger mkfastq: This is based on Illumina's bcl2fastq, which generates a fastq file for a mixed sample of sequencing samples from one or more lanes according to index tags.

cellranger count : Utilizes the fq file generated by mkfastq for comparison (STAR based), filtering, and UMI counting. Utilizes the cellbarcodeGeneration of gene-barcode matrix followed by sample clustering, gene expression analysis

cellranger aggr : accepts the output data of cellranger count and integrates the expression matrices of different sequencing samples in the same group, for example, the tumor group originally has four samples and the PBMC group has two samples, and now aggr can be used to generate the final tumor and PBMC matrices and normalize them to remove the effect of sequencing depth. of the sequencing depth

cellranger reanalyze : accepts the gene-barcode matrix generated by cellranger count or cellranger aggr, using different parameters for the(math.) lower dimensionality, clustering

The results analyzed by cell ranger software are mainly BAM, MEX, CSV, HDF5 and HTML files containing cell information.

#Download Software
curl -o cellranger-2.1. "http://cf./releases/cell-exp/cellranger-2.1.?Expires=1557260110&Policy=eyJTdGF0ZW1lbnQiOlt7IlJlc291cmNlIjoiaHR0cDovL2NmLjEweGdlbm9taWNzLmNvbS9yZWxlYXNlcy9jZWxsLWV4cC9jZWxscmFuZ2VyLTIuMS4xLnRhci5neiIsIkNvbmRpdGlvbiI6eyJEYXRlTGVzc1RoYW4iOnsiQVdTOkVwb2NoVGltZSI6MTU1NzI2MDExMH19fV19&Signature=RNQd-gTASTQhtnUSBfQWrnqo6Pyy2wDXtV5tlxkG97727GvoRhMqFXbEsz4gJl2BMckdVvW3S1tZRwRo5pmxPzmhq-8RKxf99pGqlzo84HYqhbIRkxXlIbLbj-u3PUJqo8cesWpbSVSKkS2TCNS-9GMFNieQswqMS2-DN4BqoBOJnWr7T4wlOMd9hypXWwOsW2P2fqaM-WP2ooMyo-oIxm3y9gDghXdDEP5lvHU7GCQcFGGexkdIrD6S5p8JPJ1DB5XieGrtEuP1YVp6tLMGXFoRWXS8dQLI1egWDYlOuRaiQgLIb3o5ZxBg5NpzLPP5kDHMAVzJFdBpf~~rkyNYTA__&Key-Pair-Id=APKAI7S6A5RYOXBWRPDA"
#decompression (in digital technology)
tar zxvf cellranger-2.0.

export PATH=/home/biosoft/cellranger-3.0:$PATH
echo $PATH

cellranger (2.0.2)
The result is as follows:
usage.

    cellranger mkfastq

    cellranger count
    cellranger aggr
    cellranger reanalyze
    cellranger mkloupe
    cellranger mat2csv

    cellranger mkgtf
    cellranger mkref

    cellranger vdj

    cellranger mkvdjref

    cellranger testrun
    cellranger upload
    cellranger sitecheck

We also need to download a reference genome, go to the official website to download it, this also saves us from having to use cell ranger to construct a specific reference genome, if you try to construct it yourself refer to theSingle-cell combat (three) Cell Ranger use of the first exploration。

curl -O http://cf./supp/cell-exp/refdata-cellranger-GRCh38-1.2.
# Then unzip
tar -xzvf refdata-cellranger-GRCh38-1.2.

在这里插入图片描述
CellRanger walks (IV) Overview of the Cell Ranger process Inside, you can see how to write and run the code specific to the four main functions mentioned above.

Among them.filtered_gene_bc_matrices: is an important directory, which in turn contains ,, underneath, which are the input files for downstream Seurat, Scater, Monocle, etc. analyses. This is important to note that when you go to the GEO database to download the corresponding data, it is sufficient to download to the expression matrix.

cell ranger: from library disassembly to quantificationIt's also an excellent site.

Single-cell sequencing data analysis (3) - download and use of cell ranger - study notes