RNA read alignment using transcriptome

Question

[evelyn]

Hello,

I want to align RNA reads to indexed reference transcriptome. But I could not find a way to make a transcriptome reference index and align using that with hisat2. Is the command line same for reference genome or transcriptome for hisat2.

Thank you!

Answer 1

[h.mon]

The command should be the same as for building a reference genome index. However, as far as I know, hisat2 hasn't been widely used for this, the commonly used alternatives being bwa or bowtie2.

Answer 2

[ATpoint]

Yes, the indexing is the same but you do not provide a splice site file as the transcriptome (at least this is typically the case) already contains the spliced transcripts. There is specialized software for transcriptome quantification, such as salmon and kallisto, just that you know.

[evelyn]

yes, I want to use salmon for quantification. But couldn't find specifically if I can use hisat2 for indexing the transcriptome and use it for alignment to create a bam file which can be used for salmon alignment mode. I am using:

hisat2-build transcriptome.fa transcriptome
hisat2 -q -x transcriptome -1 input_1.fastq -2 input_2.fastq -S output.sam
samtools view -bS output.sam > output.bam
salmon quant -t transcripts.fa -l -A -a output.bam -o salmon_quant

Is this approach okay. Thank you!

[Fatima]

This post might help:

A: Mapping onto the Transcriptome, RNA-seq

[ATpoint]

You do not need any alignment prior to using salmon. It will happily accept fastq files. Please read its documentation https://salmon.readthedocs.io/en/latest/salmon.html#quantifying-in-mapping-based-mode