shahcompbio/bamkallisto

Introduction

shahcompbio/bamkallisto is a bioinformatics pipeline that converts transcriptome-aligned BAM files to FASTQ and quantifies transcript expression using kallisto. It is designed for quantify expression in short-read RNAseq data against custom transcriptome references (e.g., ONT-derived assemblies) starting from existing transcriptome BAM files.

The pipeline performs the following steps:

1. Convert transcriptome BAMs to paired-end FASTQs (samtools collate/fastq)
2. Read QC (FastQC)
3. Pseudoalignment and transcript quantification (kallisto quant)
4. Gene-level summarization via tximport
5. Aggregate QC report (MultiQC)

samplesheet.csv
      |
  [parse samplesheet]
      |
  SAMTOOLS_COLLATEFASTQ (per sample)
      |
  FASTQC ──────────────────────┐
      |                        |
  KALLISTO_QUANT (per sample)  |
      |                        |
  results/{sample}/            |
    abundance.tsv              |
    abundance.h5               |
    run_info.json              |
                               |
  MULTIQC ◄────────────────────┘
      |
  multiqc_report.html

Usage

Note

If you are new to Nextflow and nf-core, please refer to this page on how to set-up Nextflow. Make sure to test your setup with -profile test before running the workflow on actual data.

First, prepare a samplesheet with your input data that looks as follows:

samplesheet.csv:

sample,transcriptome_bam
SAMPLE_1,/path/to/sample1.Aligned.toTranscriptome.out.bam
SAMPLE_2,/path/to/sample2.Aligned.toTranscriptome.out.bam

Each row represents a transcriptome-aligned BAM file for one sample.

Now, you can run the pipeline using:

# With a pre-built kallisto index
nextflow run shahcompbio/bamkallisto \
   -profile <docker/singularity> \
   --input samplesheet.csv \
   --transcripts_index /path/to/kallisto/index \
   --outdir <OUTDIR>

# With a transcriptome FASTA (index will be built automatically)
nextflow run shahcompbio/bamkallisto \
   -profile <docker/singularity> \
   --input samplesheet.csv \
   --transcripts_fasta /path/to/transcriptome.fasta \
   --outdir <OUTDIR>

# With a GTF + genome FASTA (cDNA extraction + indexing)
nextflow run shahcompbio/bamkallisto \
   -profile <docker/singularity> \
   --input samplesheet.csv \
   --gtf /path/to/annotation.gtf \
   --genome_fasta /path/to/genome.fasta \
   --outdir <OUTDIR>

Warning

Please provide pipeline parameters via the CLI or Nextflow -params-file option. Custom config files including those provided by the -c Nextflow option can be used to provide any configuration except for parameters; see docs.

For more details, see the usage documentation and the output documentation.

Parameters

Parameter	Description	Default
--input	Path to samplesheet CSV	required
--transcripts_index	Path to pre-built kallisto index	-
--transcripts_fasta	Path to transcriptome FASTA (builds index)	-
--gtf	Path to GTF annotation (used with --genome_fasta)	-
--genome_fasta	Path to genome FASTA (used with --gtf)	-
--outdir	Output directory	required
--skip_fastqc	Skip FastQC step	false
--pseudo_aligner	Pseudoaligner to use ('kallisto' or 'salmon')	kallisto
--gtf_id_attribute	Gene ID attribute in GTF	gene_id
--gtf_extra_attribute	Extra gene attribute in GTF	gene_name
--kallisto_quant_fraglen	Estimated fragment length (single-end mode)	200
--kallisto_quant_fraglen_sd	Fragment length standard deviation (single-end mode)	200
--extra_kallisto_quant_args	Extra args for kallisto (e.g., '-b 100')	-b 100

One of --transcripts_index, --transcripts_fasta, or --gtf + --genome_fasta must be provided.

Credits

shahcompbio/bamkallisto was originally written by Asher Preska Steinberg.

Contributions and Support

If you would like to contribute to this pipeline, please see the contributing guidelines.

Citations

An extensive list of references for the tools used by the pipeline can be found in the CITATIONS.md file.

This pipeline uses code and infrastructure developed and maintained by the nf-core community, reused here under the MIT license.

The nf-core framework for community-curated bioinformatics pipelines.

Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen.

Nat Biotechnol. 2020 Feb 13. doi: 10.1038/s41587-020-0439-x.