Estimating Transcript Abundance with RSEM

Question

[wes]

Dear All

I would like to run Estimating Transcript Abundance for a dataset using RSEM.

By referring to the link below, it mentioned

if alignment_based est_method:
     --aln_method <string>            bowtie|bowtie2|(path to bam file) alignment method.  (note: RSEM requires bowtie)
                                      (if you already have a bam file, you can use it here instead of rerunning bowtie)

Since I already run bowtie2 for this sample before, I would like to use the bam file for this sample instead of rerun.

May I know how to write path to bam file?

[i.sudbery]

You mention a link in your question, but there doesn't appear to be a link. How did you run bowtie2? RSEM requires that you aligned to the genome rather than to the transcriptome.

[wes]

This is the link.

https://github.com/trinityrnaseq/trinityrnaseq/wiki/Trinity-Transcript-Quantification

For bowtie2 run, the script is following the link below https://github.com/trinityrnaseq/trinityrnaseq/wiki/RNA-Seq-Read-Representation-by-Trinity-Assembly

bowtie2-build Trinity.fasta Trinity.fasta --threads 32
bowtie2 -p 10 -q --no-unal -k 20 -x Trinity.fasta -1 SRR4097977_1.trimmed.fastq.gz -2 SRR4097978_2.trimmed.fastq.gz 2>align_stats_TB2.txt| samtools view -@10 -Sb -o bowtie2_TB2.bam --threads 32

[i.sudbery]

My guess would be you do --aln_method=bowtie2_TB2.bam, but i've never quantitated using RSEM via trinity before. It you are finding it confusing, it might be worth using RSEM directly.

[srhhaines9]

I was unable to make this work. Has anyone had any success calling a previously created .bam file using --aln_method command?