This is a toy dataset consisting of ~80k segmented reads (S-reads) from a Kinnex full-length RNA library. The original HiFi reads have already been segmented using Skera, so we can directly go into primer removal using lima and such.
# Download toy S-read dataset
$ wget https://downloads.pacbcloud.com/public/dataset/IsoSeq_sandbox/human_80k_Sreads.segmented.bam
# Download the Iso-Seq v2 cDNA primers (from Iso-Seq express 2.0 kit)
$ wget https://downloads.pacbcloud.com/public/dataset/Kinnex-full-length-RNA/REF-primers/IsoSeq_v2_primers_12.fasta
$ lima --version
lima 2.9.0
$ lima human_80k_Sreads.segmented.bam IsoSeq_v2_primers_12.fasta output.bam --isoseq --peek-guess
$ ls output.*
output.IsoSeqX_bc10_5p--IsoSeqX_3p.bam
output.IsoSeqX_bc10_5p--IsoSeqX_3p.bam.pbi
output.IsoSeqX_bc10_5p--IsoSeqX_3p.consensusreadset.xml
output.json
output.lima.clips
output.lima.counts
output.lima.guess
output.lima.report
output.lima.summary
$ isoseq refine output.IsoSeqX_bc10_5p--IsoSeqX_3p.bam IsoSeq_v2_primers_12.fasta flnc.bam --require-polya
$ ls flnc.*
flnc.bam
flnc.bam.pbi
flnc.consensusreadset.xml
flnc.filter_summary.report.json
flnc.report.csv
$ isoseq cluster2 flnc.bam transcripts.bam
$ ls transcripts*
transcripts.bam
transcripts.bam.pbi
transcripts.cluster_report.csv
Multiplexed samples
This is a 12-plex regular Iso-Seq (non-Kinex) run on Sequel II system consisting of ~3 million HiFi reads.
# Download HiFi reads from a non-Kinnex (regular Iso-Seq) BAM file
$ wget https://downloads.pacbcloud.com/public/dataset/Kinnex-full-length-RNA/DATA-SQ2-UHRR-Monomer/1-CCS/m64307e_230628_025302.hifi_reads.bam
$ wget https://downloads.pacbcloud.com/public/dataset/Kinnex-full-length-RNA/DATA-SQ2-UHRR-Monomer/1-CCS/m64307e_230628_025302.hifi_reads.bam.pbi
# Download the Iso-Seq v2 cDNA primers (from Iso-Seq express 2.0 kit)
$ wget https://downloads.pacbcloud.com/public/dataset/Kinnex-full-length-RNA/REF-primers/IsoSeq_v2_primers_12.fasta
# Demux and primer removal
$ lima --isoseq --peek-guess m64307e_230628_025302.hifi_reads.bam IsoSeq_v2_primers_12.fasta output.bam
# Combine inputs
$ ls output.IsoSeqX*bam > all.fofn
# Remove poly(A) tails and concatemer
$ isoseq refine all.fofn IsoSeq_v2_primers_12.fasta flnc.bam --require-polya
$ isoseq cluster2 flnc.bam transcripts.bam