Real-world example

This is an example of an end-to-end cmd-line-only workflow:

# Download HiFi reads
$ wget https://downloads.pacbcloud.com/public/dataset/IsoSeq_sandbox/2022_pbmc_singlecell_mini/ccs.bam
$ wget https://downloads.pacbcloud.com/public/dataset/IsoSeq_sandbox/2022_pbmc_singlecell_mini/ccs.bam.pbi

# Download cDNA primers
$ wget https://downloads.pacbcloud.com/public/dataset/IsoSeq_sandbox/2022_pbmc_singlecell_mini/primers.fasta

# Download cell barcode include list
$ wget https://downloads.pacbcloud.com/public/dataset/IsoSeq_sandbox/10x_barcodes/3M-february-2018-REVERSE-COMPLEMENTED.txt.gz

# Check lima version to be >= 2.6.0
$ lima --version
lima 2.6.0

# Check isoseq version to be >= 4.0.0
$ isoseq --version
isoseq 4.0.0 

# cDNA primer removal and read orientation
$ lima --per-read --isoseq ccs.bam primers.fasta output.bam

# Clip UMI and cell barcode
$ isoseq tag output.5p--3p.bam flt.bam --design T-12U-16B

# Remove poly(A) tails and concatemer
$ isoseq refine flt.bam primers.fasta fltnc.bam --require-polya

# Correct single cell barcodes based on an include list
$ isoseq correct -B 3M-february-2018-REVERSE-COMPLEMENTED.txt.gz fltnc.bam corrected.bam

# Deduplicate reads based on UMIs
$ samtools sort -t CB corrected.bam -o corrected.sorted.bam
$ isoseq groupdedup corrected.sorted.bam dedup.bam

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