NGS piplelines

  • Quality Control
    • Previous QC from DNA lectures
  • Alignment
    • Alignment without splicing (DNA)
    • with splicing (RNA)
  • Expression level estimate and comparison
  • Tophat and cufflinks in Nature Methods DOI: 10.1038/nprot.2012.016

Presenter Notes

Alignment with Bowtie and TopHat

BowTie

A fast aligner for short reads. This is comparable to bwa but previous versions were shown to be less sensitive.

Requires genome file to be indexed just like bwa does.

$ bowtie-build genome.fa genome
# or (better to try and use bowtie2 which is faster)
$ bowtie2-build genome.fa genome

Basis for the alignments used in TopHat which supports spliced alignment.

$ tophat genome -G genome_annotation.gtf rnaseq_1.fq rnaseq_2.fq

Presenter Notes

mRNA alignment

How to align when the short reads may be interrupted by introns in the genome? Spliced alignment can be made aware of potential sites

tophat1

Presenter Notes

splicing approaches

nmeth.1613-F1

Cufflinks

For comparing expression values. This will generate table of expression values per transcript and gene unit.

$ cufflinks -G genome.gff -I MAX_INTRON_SIZE --library-type [fr-unstranded] tophat/accepted_hits.bam

Presenter Notes

Denovo annotation with TopHat/Cufflinks

Cufflinks will generate transcript models de-novo from annotation.

cufflinks

Presenter Notes

Transcript assembly with Trinity

Trinity Trinity

Presenter Notes

Trinity on biocluster

$ module load trinity-rnaseq
$ ulimit -s unlimited
$ Trinity.pl --max_memory 30G --CPU  20 --kmer_method inchworm \   
  --seqType fq --left Neo_RNA_seq-RNA_l1_1.fq \
  --right Neo_RNA_seq-RNA_l1_2.fq --output trinity_out \
  --jaccard_clip

Presenter Notes