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Quantification in Alignment mode for Nanopore Data #903
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Harshangda
changed the title
Quantifyication in Alignment mode for Nanopore Data
Quantification in Alignment mode for Nanopore Data
Dec 8, 2023
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. |
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
…________________________________
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.
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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.
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
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