RNAseq教程(1.2)

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

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。

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

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)

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?

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)?

cat chr22_with_ERCC92.fa | grep -v ">" | perl -ne 'chomp $_; $bases{$_}++ for split //; if (eof){print "$_ $bases{$_}\n" for sort keys %bases}'

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

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 = $r\ntotal bases = $l\npercent repeat bases = $p%\n\n"}'

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。

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 = $c\n\n";}'
EcoRI site (GAATTC) count = 3935