Quantification in Alignment mode for Nanopore Data

[Harshangda]

I am using Salmon (Salmon 1.3 installed through homebrew on mac ventura 13.5.2) for quantification in alignment mode. I have bam files (aligned to transcriptome) for nanopore data from Minimap2, which has primary and secondary alignments both (50% of total mapped reads are primary and other 50% are secondary alignments). Salmon is only performing quantification for primary alignments, but I also want to include the secondary alignments.

1. Is there any argument I could use to include secondary alignments for quantification in Salmon Alignment mode?
2. Is it a good practice to include secondary alignments? 3. Are there any other important flags I should use which might be specific to "Nanopore" or "Splicing Analysis".

This is the code I am using at the moment:

salmon quant -t gencode.vM33.transcripts.fa -l A -g gencode.vM32.annotation.gtf -a barcode07.bam -o barcode_07_salmon_quant

[alexyfyf]

also interested to know how Salmon uses secondary alignment. Because I found this tutorial https://combine-lab.github.io/salmon-tutorials/2021/ont-long-read-quantification/ actually includes secondary alignments. And based on my experience, secondary alignments are used by Salmon, because when I give a BAM before and after removing secondary (-F 256 flag in samtools), the results are different.

[Harshangda]

Hi,

If I don't trim the adaptors and still use --ont will I still get correct quantification? Is adaptor trimming very essential? Is there a way I can use salmon without adaptor trimming?

Also, can you please clarify about the secondary alignmenmts if these are included in Salmon or not?

Thanks, Harsha

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From: Feng Yan @.> Sent: 08 January 2024 23:30 To: COMBINE-lab/salmon @.> Cc: Harshangda Karan Puri @.>; Author @.> Subject: Re: [COMBINE-lab/salmon] Quantification in Alignment mode for Nanopore Data (Issue #903)

also interested to know how Salmon uses secondary alignment. Because I found this tutorial https://combine-lab.github.io/salmon-tutorials/2021/ont-long-read-quantification/ [combine-lab.github.io]https://urldefense.com/v3/__https://combine-lab.github.io/salmon-tutorials/2021/ont-long-read-quantification/__;!!PDiH4ENfjr2_Jw!GTZeAEdMSJcSBTPXhWuSsmLuX2WDzuNuNgqT04lADpRqOWyHssr_JALdqVa1JBOS9RHGRa9M6SeJKoxo6T7o5_O0bvsV-KkgNb45i4uTnGob8fw$ actually includes secondary alignments. And based on my experience, secondary alignments are used by Salmon, because when I give a BAM before and after removing secondary (-F 256 flag in samtools), the results are different.

— Reply to this email directly, view it on GitHub [github.com]https://urldefense.com/v3/__https://github.com/COMBINE-lab/salmon/issues/903*issuecomment-1881982972__;Iw!!PDiH4ENfjr2_Jw!GTZeAEdMSJcSBTPXhWuSsmLuX2WDzuNuNgqT04lADpRqOWyHssr_JALdqVa1JBOS9RHGRa9M6SeJKoxo6T7o5_O0bvsV-KkgNb45i4uTEiG0xQE$, or unsubscribe [github.com]https://urldefense.com/v3/__https://github.com/notifications/unsubscribe-auth/A3SZAPCLOZYB72ZEIEEXH43YNR6S7AVCNFSM6AAAAABANBCPNSVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMYTQOBRHE4DEOJXGI__;!!PDiH4ENfjr2_Jw!GTZeAEdMSJcSBTPXhWuSsmLuX2WDzuNuNgqT04lADpRqOWyHssr_JALdqVa1JBOS9RHGRa9M6SeJKoxo6T7o5_O0bvsV-KkgNb45i4uTntkMlxE$. You are receiving this because you authored the thread.Message ID: @.***>