目錄

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

1.4 Indexing

創(chuàng)建HISAT索引

為chr22和ERCC spikein序列創(chuàng)建HISAT2索引。HISAT2可以將外顯子和剪接位點(diǎn)合并到索引文件中進(jìn)行對(duì)齊。首先創(chuàng)建一個(gè)剪接站點(diǎn)文件，然后創(chuàng)建一個(gè)外顯子文件。最后制作FM索引。

要了解更多關(guān)于HISAT2索引策略與其他軟件的不同之處，請(qǐng)參閱說(shuō)明書(shū)。

hisat2_extract_splice_sites.py chr22_with_ERCC92.gtf >INDEX/splicesites.tsv
hisat2_extract_exons.py chr22_with_ERCC92.gtf >INDEX/exons.tsv
hisat2-build -p 8 --ss INDEX/splicesites.tsv --exon INDEX/exons.tsv chr22_with_ERCC92.fa INDEX/index

[可選]為所有染色體創(chuàng)建索引，而不是僅僅為chr22，你會(huì)做以下操作:

注意:下面的例子沒(méi)有利用將剪接位點(diǎn)和外顯子添加到索引。如果需要，可以使用完整的GTF生成這些文件，并使用適當(dāng)?shù)倪x項(xiàng)將它們添加到命令中。

警告:為了索引整個(gè)人類基因組，HISAT2需要160GB內(nèi)存。AWS實(shí)例大小將耗盡RAM。

#cd /home/ubuntu/workspace/data/fasta/GRCh38
#hisat2-build -p 8 Homo_sapiens.GRCh38.dna_sm.primary_assembly.fa Homo_sapiens.GRCh38.dna_sm.primary_assembly