Demultiplexing using lima

[Nitin123-4]

Hi team I have: Kinnex run bam, these are the segmented reads in bam file.

For demultiplexing, the barcoded cDNA amplification primers we used are:

SAMP102-001:

IsoSeqX_bc01_5p CTACACGACGCTCTTCCGATCTACTACACGCAATGAAGTCGCAGGGTTGGG IsoSeqX_3p AAGCAGTGGTATCAACGCAGAGTAC

SAMP102-002:

IsoSeqX_bc02_5p CTACACGACGCTCTTCCGATCTACTAGTAGCAATGAAGTCGCAGGGTTGGG IsoSeqX_3p AAGCAGTGGTATCAACGCAGAGTAC

For demultiplexing can I use:

lima --isoseq --dump-clips --peek-guess -j 24 FILE-segmentedreads.bam SAMP102-001.cDNA_amplification_primers.fasta SAMP102-001.demult.bam lima --isoseq --dump-clips --peek-guess -j 24 FILE-segmentedreads.bam SAMP102-002.cDNA_amplification_primers.fasta SAMP102-002.demult.bam

OR I need to create one _primers.fasta where I can give info as:

SAMP102-001_5p CTACACGACGCTCTTCCGATCTACTACACGCAATGAAGTCGCAGGGTTGGG SAMP102-002_5p CTACACGACGCTCTTCCGATCTACTAGTAGCAATGAAGTCGCAGGGTTGGG primer_3p AAGCAGTGGTATCAACGCAGAGTAC

and then run:

lima --isoseq --dump-clips --peek-guess -j 24 FILE-segmentedreads.bam primers.demux.fasta

Please suggest.

[armintoepfer]

You combine them and run demux once

[Nitin123-4]

When I combine them and run I can see reads are distributed for both of them, but when I am doing it one at a time looks like almost all the reads are going in both the samples.

Thanks.