2020-06-21

单词

established adj.已确立的；已获确认的；确定的；

Analysis of single cell RNA-seq data

1. download all necessary files(bash poststart.sh)
   Or selective download：link

# poststart.sh
aws --endpoint-url https://cog.sanger.ac.uk --no-sign-request s3 cp s3://scrnaseq-course/data data --recursive

2. download fastq files(link)

# 查看文件
less Share/ERR522959_1.fastq
less Share/ERR522959_2.fastq

3. FastQC

mkdir fastqc_results
fastqc -o fastqc_results Share/ERR522959_1.fastq Share/ERR522959_2.fastq

4. Trimming Reads

mkdir fastqc_trimmed_results
# --nextera是由 FastQC报告中的‘Adaptor Content’ 决定的
trim_galore --nextera -o fastqc_trimmed_results Share/ERR522959_1.fastq Share/ERR522959_2.fastq

5. Using STAR to Align Reads

mkdir indices
mkdir indices/STAR

mkdir results
mkdir results/STAR

# setp1: create the index
STAR --runThreadN 4 --runMode genomeGenerate --genomeDir indices/STAR --genomeFastaFiles Share/2000_reference.transcripts.fa

# setp2: perform the mapping step
# 报错未解决: EXITING because of FATAL ERROR in reads input: short read sequence line: 0
# Read Name=@ERR522959.4784336
# Read Sequence====
# DEF_readNameLengthMax=50000
# DEF_readSeqLengthMax=650
STAR --runThreadN 4 --genomeDir indices/STAR --readFilesIn fastqc_trimmed_results/ERR522959_1_trimmed.fq fastqc_trimmed_results/ERR522959_2_trimmed.fq --outFileNamePrefix results/STAR/


STAR --runThreadN 4 --genomeDir indices/STAR --readFilesIn Share/ERR522959_1.fastq Share/ERR522959_2.fastq --outFileNamePrefix results/STAR/

6. Kallisto and Pseudo-Alignment

• STAR is a reads aligner, whereas Kallisto is a pseudo-aligner. The main difference between aligners and pseudo-aligners is that whereas aligners map reads to a reference, pseudo-aligners map k-mers to a reference.
• Unlike STAR, Kallisto psuedo-aligns to a reference transcriptome rather than a reference genome.
• batch.txt文件创建参考https://pachterlab.github.io/kallisto/manual
  创建batch.txt文件，输入以下内容：
  #id file1 file2
  cell1 Share/ERR522959_1.fastq Share/ERR522959_2.fastq

mkdir indices/Kallisto
mkdir results/Kallisto

# step1: produce the Kallisto index
kallisto index -i indices/Kallisto/transcripts.idx Share/2000_reference.transcripts.fa

# step2:  perform pseudo-alignment
kallisto pseudo -i indices/Kallisto/transcripts.idx -o results/Kallisto -b batch.txt 

7. Construction of expression matrix

R

1. R packages can be downloaded and installed directly from github using the “devtools” package.

install.packages("devtools")
require("devtools")
# the most recent “master” version of the package
devtools::install_github("tallulandrews/M3Drop")
# different branch
devtools::install_github("tallulandrews/M3D", ref="nbumi")
# previous commit
devtools::install_github("tallulandrews/M3Drop", ref="434d2da28254acc8de4940c1dc3907ac72973135")