单细胞分析pipeline设计与验证

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1.软件安装
cell ranger 6.1

curl -o cellranger-6.1.0.tar.gz "https://cf.10xgenomics.com/releases/cell-exp/cellranger-6.1.0.tar.gz?Expires=1628798968&Policy=eyJTdGF0ZW1lbnQiOlt7IlJlc291cmNlIjoiaHR0cHM6Ly9jZi4xMHhnZW5vbWljcy5jb20vcmVsZWFzZXMvY2VsbC1leHAvY2VsbHJhbmdlci02LjEuMC50YXIuZ3oiLCJDb25kaXRpb24iOnsiRGF0ZUxlc3NUaGFuIjp7IkFXUzpFcG9jaFRpbWUiOjE2Mjg3OTg5Njh9fX1dfQ__&Signature=IDnv8TCqfkxaEFEt3c6rwO~dWltlWa5Kr5b-CQeOgGMFjmVYwWVCLZ1pEKnoncD8waXaUj9gCNIChyDjS-nETYLwSEK~n1JkEUkjYZwLhPjKWlG87BEIjT1WORM4SotaaC2Vys5QvgBZh1YOh5VqqEJygpUFRMd0dNHTi0BXPFVk44iGhb~qXwvjIIpfogcbljZ~ytF5AsQv387VL4qRvlmhsGkApaNmWLwsBD9RdEwLEbQ3z4QaBCtI9lKHqduU-TF-b4~HNutYuniNWlVf9NjrdJngsYTS-0fY-CoKQeJb9ZZ8S5n6~wKsPdAtjrxfWKOEXAdmrw5-HHySE7fVuQ__&Key-Pair-Id=APKAI7S6A5RYOXBWRPDA"
$ cd /opt
[ download file from downloads page ]
$ tar -xzvf cellranger-6.1.0.tar.gz

下载参考基因组

curl -O https://cf.10xgenomics.com/supp/cell-exp/refdata-gex-GRCh38-2020-A.tar.gz
$ tar -xzvf refdata-gex-GRCh38-2020-A.tar.gz

下载bcl2fastq
https://support.illumina.com/downloads/bcl2fastq-conversion-software-v2-20.html

bcl2fastq_letterbooklet_15038058brpmi.pdf
进入 src
./configure & make & make install

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n file included from /ceph_disk3/single_cell/bcl2fastq/src/cxx/include/common/Debug.hh:22:0,
from /ceph_disk3/single_cell/bcl2fastq/src/cxx/lib/common/InstallationPath.cpp:22:
/ceph_disk3/single_cell/bcl2fastq/src/cxx/lib/common/InstallationPath.cpp: In constructor ‘bcl2fastq::common::InstallationPath::InstallationPath()’:
/ceph_disk3/single_cell/bcl2fastq/src/cxx/include/common/Logger.hh:38:14: error: ‘clog’ is not a member of ‘std’

vi /ceph_disk3/single_cell/bcl2fastq/src/cxx/include/common/Logger.hh

在头文件添加 #include <iostream>

/ceph_disk3/single_cell/bcl2fastq/src/cxx/lib/io/Xml.cpp:175:104: error: no matching function for call to ‘xml_writer_make_settings(char, int)’
boost::property_tree::write_xml(os, tree, boost::property_tree::xml_writer_make_settings(' ', 2));
https://gist.github.com/moonwatcher/ac1176971d6032fc9e0d4402e5a81333

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cellranger mkfastq --help

There are three arguments or inputs that are added to the cellranger mkfastq command: –-id, --run, and --csv.

The --id can be anything. It is used by the pipeline to name the output directory that Cell Ranger is going to create to run in. This directory is called a pipestance, which is short for pipeline instance.

The --run argument points to the Illumina run folder that contains the BCL files.

The --csv argument is a comma-separated values (CSV) file that describes how samples were indexed on the Illumina flow cell.

cellranger mkfastq --id=tutorial_walk_through \
--run=/mnt/home/user.name/yard/run_cellrnager_mkfastq/cellranger-tiny-bcl-1.2.0 \
--csv=/mnt/home/user.name/yard/run_cellrnager_mkfastq/cellranger-tiny-bcl-simple-1.2.0.csv

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计算 count

wget https://cf.10xgenomics.com/samples/cell-exp/3.0.0/pbmc_1k_v3/pbmc_1k_v3_fastqs.tar

--id 会根据这个id创建运行临时输出目录

output有多种输出格式

咱们比较适合用原始的矩阵
https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/output/matrices

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https://bioinformatics.stackexchange.com/questions/4271/what-is-cellranger-doing-in-comparison-to-other-methods

cell ranger 其实是一个pipeline 内部也调用了 star 这些软件我们其实可以把这个pipeline 打开做成一个通用而且可以调整的流程类似于DropSeq
考虑到 cell ranger 只能支持10X的实验数据大家已经意识到这个问题了未来估计会出现一个通用的流程：
https://www.biorxiv.org/content/10.1101/2020.02.09.940221v1.full