RNAseq 1.2

2021-09-07 18:11:51 浏览数 (1)

目录

1.Module 1 - Introduction to RNA sequencing

  1. Installation
  2. Reference Genomes
  3. Annotations
  4. Indexing
  5. RNA-seq Data
  6. Pre-Alignment QC

2.Module 2 - RNA-seq Alignment and Visualization

  1. Adapter Trim
  2. Alignment
  3. IGV
  4. Alignment Visualization
  5. Alignment QC

3.Module 3 - Expression and Differential Expression

  1. Expression
  2. Differential Expression
  3. DE Visualization
  4. Kallisto for Reference-Free Abundance Estimation

4.Module 4 - Isoform Discovery and Alternative Expression

  1. Reference Guided Transcript Assembly
  2. de novo Transcript Assembly
  3. Transcript Assembly Merge
  4. Differential Splicing
  5. Splicing Visualization

5.Module 5 - De novo transcript reconstruction

  1. De novo RNA-Seq Assembly and Analysis Using Trinity

6.Module 6 - Functional Annotation of Transcripts

  1. Functional Annotation of Assembled Transcripts Using Trinotate

1.2 Reference Genomes

从Ensembl、iGenomes、NCBI或UCSC获得参考基因组。在本例分析中，我们将使用人GRCh38版本的Ensembl基因组。此外，我们实际上将只使用单个染色体(chr22)和ERCC spikein来执行分析，以使它运行得更快……

创建必要的工作目录

代码语言：javascript复制
mkdir RNA_ref

这些s数据可以在ftp://ftp.ensembl.org/pub/release-86/fasta/homo_sapiens/dna/找到。你可以使用wget下载homo_sapien . grch38 .dna_sm.primary_assembly.fa.gz文件，然后解压缩/解压。

代码语言：javascript复制
cd RNA_ref
wget http://genomedata.org/rnaseq-tutorial/fasta/GRCh38/chr22_with_ERCC92.fa
ls

查看该文件的前10行。为什么会是这个样子

代码语言：javascript复制
head chr22_with_ERCC92.fa

这个文件中有多少行和字符?这条染色体有多长(碱基和Mbp)

代码语言：javascript复制
wc chr22_with_ERCC92.fa
 848761   848764 51751056 chr22_with_ERCC92.fa

查看大约从该文件中间开始的10行。大小写字符的意义是什么?

代码语言：javascript复制
head -n 425000 chr22_with_ERCC92.fa | tail
ggaggctgaggcaggagaatcgcttgaacatgggaggtggaagttgcagtgagccgaaac
tgcgccattgcactatagcctgggcaacaagagtgaaagtctgtcttgaaaaaaaaaaaT
CAGATGTTCTATGTAAAAATGCTATCTAtgattgaagtataaaactttacctccctttat
gttcctttgccctccccactatttattattgtcttgattatatcttctatatgcattgag
aggtgttataacttttgtatcaatcaccaaatttaatttagaaaatataagaggagaaga
aaagtctattacatttactcatatttttgcttactgtgttctttcttccttcttgatgtt
ccagaatttcttttattgcttcttttctgcttagaaaactttatctttttctttcatctt
tcttttttcctcctcctcctcctcctcctttttttttttttttttttttttttttttaat
aaagagacagggtctcactctatcacccagactggagttcagtgatgcaatcatagctca
ttgcaaccttgaactcctgggctcaagtgatcctcccacctcagcctcctgagtagctgg

在整个参考基因组文件中每个碱基的计数是多少(跳过每个序列的标题行)?

代码语言：javascript复制
cat chr22_with_ERCC92.fa | grep -v ">" | perl -ne 'chomp $_; $bases{$_}   for split //; if (eof){print "$_ $bases{$_}n" for sort keys �ses}'

A 4455938
C 4406493
G 4411768
N 10710000
T 4445994
Y 1
a 5950524
c 4772185
g 4853055
n 948691
t 5946575

请记住引用序列(染色体)的名称必须与注释gtf文件(在下一节中描述)中匹配。

练习2

22号染色体上有多少个碱基对应于重复的元素?整个长度占的百分比是多少

代码语言：javascript复制
cat chr22_with_ERCC92.fa | perl -ne 'if ($_ =~ />22/){$chr22=1}; if ($_ =~ />ERCC/){$chr22=0}; if ($chr22){print "$_";}' > chr22_only.fa
cat chr22_only.fa | grep -v ">" | perl -ne 'chomp $_; $r = $_ =~ tr/a/A/; $r  = $_ =~ tr/c/C/; $r  = $_ =~ tr/g/G/; $r  = $_ =~ tr/t/T/; $l  = length($_); if (eof){$p = sprintf("%.2f", ($r/$l)*100); print "nrepeat bases = $rntotal bases = $lnpercent repeat bases = $p%nn"}'

repeat bases = 21522339
total bases = 50818468
percent repeat bases = 42.35%

22号染色体序列中出现多少个EcoRI限制位点?EcoRI限制性内切酶识别序列为5'- GAATTC -'3。

代码语言：javascript复制
cat chr22_only.fa | grep -v ">" | perl -ne 'chomp $_; $s = uc($_); print $_;' | perl -ne '$c  = $_ =~ s/GAATTC/XXXXXX/g; if (eof){print "nEcoRI site (GAATTC) count = $cnn";}'
EcoRI site (GAATTC) count = 3935
express 编程算法

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