salmon light weight alignment mode

Question

[evelyn]

Hello,

I have sorted bam files for paired end RNA seq fastq files using Hisat2, Picard and I want to use salmon alignment mode:

hisat2 -q -x genome -1 input_1.fastq -2 input_2.fastq -S out.sam
samtools view -bS out.sam > out.bam
samtools sort out.bam -o out.sorted.bam
samtools index out.sorted.bam

java -jar picard.jar MarkDuplicates \
I=out.sorted.bam \
O=out_marked_duplicates.bam \
java -jar picard.jar BuildBamIndex \ INPUT=out_marked_duplicates.bam

Then to use salmon,

salmon quant -t transcripts.fa -l -A -a out_marked_duplicates.bam -o salmon_quant

I am not sure if there is any different option for using this sorted bam file for salmon. I will appreciate any comments/help! Thank you!

[ATpoint]

Everything you need to know is listed in the documentation: https://salmon.readthedocs.io/en/latest/salmon.html#quantifying-in-alignment-based-mode

I do not see any specific question, is there any? Also, why aligning data if you end up using salmon? salmon can use fastq rigth away.

[evelyn]

Thank you! I want to see any difference using this approach as compared to using fastq files. I could not find an option for light weight alignment in the documentation.

[lieven.sterck]

as a side note: I would not blindly remove duplicates if your goal is expression analysis. It might bias your analysis.

[evelyn]

I am not going to remove the duplicates. I am looking for a way to use salmon alignment for sorted bam files created with hisat2 including the reference genome.