单细胞：CEL-Seq2 数据分析（劝退篇）

参考文献：CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq

github：GitHub - yanailab/CEL-Seq-pipeline

下载：

wget https://github.com/yanailab/CEL-Seq-pipeline/archive/refs/tags/v1.0.tar.gz

解压后只有这些文件

CEL-Seq-pipeline-1.0/
├── bc_demultiplex.py
├── bowtie_wrapper.py
├── clean_up.py
├── htseq_count_umified.py
├── htseq_wrapper.py
├── LICENSE
├── pijpleiding.py
└── README.md

使用方法：

pijpleiding config_file.txt

准备工作：

• 你的fastq文件，
• barcode index文件（barcode_umis.tab)
• sample_sheet txt文件 (sample_sheet_example.txt)

安装软件：python2， hiseq(失败)

python2 -m pip  install HTSeq

image.png

HTSeq已经不支持python2了：

image.png

如果你有python2 的HTSeq包，可以继续：

首先创建一个config_file.txt，修改里面的参数和路径。

##  pijpleiding configuration file. Run `pijplieding --help` for more help.
##
##  the pipe_run parameter decides whether to run the pipe segment or not.
##  the pipe_input_files (which can be multilined) is treated as multiple shell
##  patterns refering to existing files, so it is expanded and split accordingly,
##  and passed as 'input_files' to the pipe segment. The rest of the parameters
##  are passed as they are to the pipe segments, so check their description

[scythe_wrapper]
pipe_run = True

[bc_demultiplex]
pipe_run = True

bc_index_file= /path_to/barcodes_umis.tab
sample_sheet= /path_to/Sample_sheet.txt
pipe_input_files= /path_to/*/*R1*.fastq
output_dir= /path_to/barcode_splitted
stats_file= stats.tab
min_bc_quality= 10
bc_length = 6
umi_length = 5
cut_length = 35

[bowtie_wrapper]
pipe_run = True

pipe_input_files= /path_to/barcode_splitted/CE_*.fastq
index_file= /path_to/refs/genomes/CE/WS230/c_elegans.WS230_spikein.genomic
output_dir= /path_to/sam_files
bowtie_report_name = bt_report_full.tab
number_of_threads = 3
extra_params =
procs = 10

[htseq_wrapper]
pipe_run = True

pipe_input_files = /path_to/sam_files/*sam
gff_file = /path_to/refs/annotations/CE/WS230/c_elegans.WS230_spikein.annotations_trimmed.spikes_and_lincs.gff3
output_dir= /path_to/expression_umi
umi= true
extra_params = -q
count_filename = CE_exp.tab

[clean_up]
pipe_run = False

最后使用：

python2 CEL-Seq-pipeline-1.0/pijpleiding.py config.txt

总结： 这么过时的代码，就别用了吧！