Contents

0.1 Cleaning up data
1 Building genome with HISAT2
- 1.1 GTFs:
- 1.2 Using HISAT2 and related software on server:
2 Building your annotated genome for mapping with HISAT2.
3 Alignment with HISAT2
- 3.1 Note on --rna-strandness
- 3.2 Intermediate file conversions
4 StringTie for assembling transcripts
- 4.1 StringTie for merging transcripts
- 4.2 gffcompare to compare to annotations:
- 4.3 Calculate transcript abundances with stringtie for use with ballgown or other DE analysis software.
5 In class exercise 1:
6 Counting reads in transcripts with HTseq
- 6.1 Important update on HTseq-count
- 6.2 Strandedness with HTSeq
7 In class exercise 2:
8 Use Genome Coverage from bedtools to create a bedGraph:
- 8.1 Note on visualizing stranded data with genomeCoverageBed
9 In class exercise 3:
10 Processing paired end RNA-seq and visualizing splicing
11 Visualizing stranded PE data:
- 11.1 Mapping paired end and stranded data with HISAT2
12 Displaying splice junctions
- 12.1 Move header to a new file
- 12.2 Extract splice junction reads
- 12.3 Convert back to bam
- 12.4 Convert to bed12
13 Making bedGraphs files of C_RNAseq_chr17_pe.bed12.bed data above (all split spliced reads and non-split reads).
14 Exercise 4: Extract and visualize splice junctions from paired end RNA-seq data.
15 For your reference: Molecular biology of library preparations
16 Answers to in class exercise 1:
17 Answers to class exercise 2:
18 Answers to in class exercise 3:
19 Answers to in class exercise 4: