NGS expression analysis¶
There are several interesting RNA-Seq reviews: From RNA-seq reads to differential expression results, Microarrays, deep sequencing and the true measure of the transcriptome, Computational methods for transcriptome annotation and quantification using RNA-seq, Next-generation transcriptome assembly, An opinionated guide to the proper care and feeding of your transcriptome and Evaluation of de novo transcriptome assemblies from RNA-Seq data.
There are also some interesting studies regarding the post-processing, quality of the assembled transcriptomes, the alternative transcript reconstruction success of different algorithms, the evaluation of different assembly strategies.
StringTie reconstruction algorithm.
transrate, a software to evaluate the transcriptome assembly quality.
Mapping and count sequences¶
First we create an index of genome file.
ngs_user@machine:~/rnaseq$ mkdir rnaseq ngs_user@machine:~/rnaseq$ bowtie2-build sl2.fasta sl2
Now, we map the reads.
ngs_user@machine:~/rnaseq$ tophat sl2 SRR45_region.fastq -o SRR45 ngs_user@machine:~/rnaseq$ less ./SRR45/align_summary.txt
We sort and index the bam file to open with IGV.
ngs_user@machine:~/rnaseq$ samtools sort ./SRR45/accepted_hits.bam ./SSR45/accepted_hits_45.sort ngs_user@machine:~/rnaseq$ samtools index ./SSR45/accepted_hits_45.sort
Now, we can construct the transcripts and the number of reads in each transcript. We need also the
genome annotation file
ngs_user@machine:~/rnaseq$ cufflinks -g sl2.gff3 ./SRR45/accepted_hits.bam ngs_user@machine:~/rnaseq$ ls ngs_user@machine:~/rnaseq$ less transcripts.gtf ngs_user@machine:~/rnaseq$ less genes.fpkm_tracking
Load the accepted_hits_45.sort.bam file, the transcripts.gtf and the annotation file sl2.gff3 on the IGV to test the mapping and transcript assembly done with Cufflinks. The next steps will be integrate all transcript assemblies of all samples to create a new annotation file and use it to quantify again the reads of each sample. After that we will start the normalization, differential analysis, etc.