IsoSeq-Nextflow

Nextflow implementation of IsoSeq3

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Steps

The IsoSeq pipeline is implemented in the following steps

1. lima
   • demultiplexing and primer removal
2. merge
   • combine ccs reads based on barcode pair
   • only valid barcodes pairs are merged - all combinations of 3p__5p barcode pairs as defined in barcodes.fasta
3. refine
   • polyA tail trimming
   • concatemer removal
4. cluster
   • hierarchical, n*log(n) clusttering, alignment of shorter to longer sequences
   • iterative cluster merging
   • generate consensus for each read cluster using QV guided PoA
5. align
   • align reads to genome using pbmm2
6. collapse
   • collapse reads

Running the Pipeline with Docker

nextflow run isoseq3.nf --ccs_reads ccs/read/dir --barcodes barcodes.fasta --genome_fasta genome.fasta --name example

Running the Pipeline with Singularity

nextflow run isoseq3.nf --ccs_reads ccs/read/dir --barcodes barcode.fasta --genome_fasta genome.fasta --name example -with-singularity -without-docker

Input

1. --ccs_reads
   • ccs.bam file or directory containing ccs.bam files
2. --barcodes
   • barcode primers to use when demultiplexing
   • 3' barcodes must end in _3p
   • 5' barcodes must end in _5p
3. --genome_fasta
   • genome fasta file to use in alignment
4. --name
   • name of experiment