Strategies for Bulk RNA-seq Analysis Genome Transcriptome - - PowerPoint PPT Presentation

Strategies for Bulk RNA-seq Analysis

Genome Mapping Transcriptome Mapping Assembly

Reads Reads Reads Transcript mapping and quantification Splice-aware Genome mapping RSEM, Kallisto, Sailfish, Salmon STAR, HISAT2 Gene counting Transcript discovery & counting htseq-count, featureCounts StringTie Novel transcript annotation Homology-based BLAST2GO Assembly into transcripts Trinity, Scripture, Stringtie Novel transcript annotation Trinotate

multiple BAMs (+known GTF)

Sequence reads Quality control Alignment to Genome: HISAT2, STAR DGE with R: DESeq2, EdgeR, limma:voom Count reads associated with genes: htseq-count, featureCounts

FASTQ FASTQ Count Matrix (+known GTF, optional) (+reference genome index)

✓ Genome ✓ GTF (annotation)

multiple BAMs (+known GTF)

Sequence reads Quality control Alignment to Genome: HISAT2, STAR DGE with R: DESeq2, EdgeR, limma:voom Count reads associated with genes: htseq-count, featureCounts Reference-based transcriptome assembly and quantitation with StringTie

FASTQ FASTQ Count Matrix (+known GTF, optional) BAM (+reference genome index)

✓ Genome ✓ GTF (annotation)?

DGE with CuffDiff, Ballgown

(+reference genome index) multiple BAMs (+known GTF)

Sequence reads Quality control Alignment to Genome: HISAT2, STAR DGE with R: DESeq2, EdgeR, limma:voom Count reads associated with genes: htseq-count, featureCounts Reference-based transcriptome assembly and quantitation with StringTie

FASTQ FASTQ Count Matrix BAM

✓ Transcriptome

(FASTA)

DGE with CuffDiff, Ballgown

(nown GTF, optional) (+reference transcriptome index) FASTQ

DGE with Sleuth Pseudocounts with Kallisto, Sailfish, Salmon

Count Matrix generated using tximport

Reference-based assembly De novo assembly

Martin J.A. and Wang Z., Nat. Rev. Genet. (2011) 12:671–682

✓ Genome ✓ GTF (annotation)? ✓ Genome? ✓ GTF (annotation)?

Quantitation from assembled reads

Alignment to new transcriptome: Bowtie2, BWA DGE with R: DESeq2, EdgeR, limma:voom Count reads associated with genes

Count Matrix SAM/BAM

Reads Assembly into transcripts Trinity, Scripture RSEM, Kallisto. Salmon, eXpress Transcript mapping & quantification Novel transcript annotation Trinotate

These materials have been developed by members of the teaching team at the Harvard Chan Bioinformatics Core (HBC). These are open access materials distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.