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Basic workflow for nanopore sequencing data

License: MIT License

Nextflow 73.92% Shell 7.40% Python 18.68%

bioinformatics nanopore denovo-assembly polishing nextflow

donut_falls's Introduction

Donut Falls

Named after the beautiful Donut Falls

Location: 40.630°N 111.655°W, Elevation: 7,942 ft (2,421 m), Hiking level: easy

(Image credit: User submitted photos at alltrails.com)

More information about the trail leading up to this landmark can be found at utah.com/hiking/donut-falls

Donut Falls is a Nextflow workflow developed by @erinyoung at the Utah Public Health Laborotory for long-read nanopore sequencing of microbial isolates. Built to work on linux-based operating systems. Additional config options are needed for cloud batch usage.

Donut Falls is also included in the staphb toolkit staphb-toolkit.

We made a wiki, please read it!

Wiki table of contents:

Getting started

Install dependencies

Nextflow
Singularity or Docker

Quick start

nextflow run UPHL-BioNGS/Donut_Falls -profile <singularity or docker> --sample_sheet <sample_sheet.csv>

Sample Sheets

Sample sheet is a csv file with the name of the sample and corresponding nanopore fastq.gz file on a single row with header sample and fastq. When Illumina fastq files are available for polishing or hybrid assembly, they are added to end of each row under column header fastq_1 and fastq_2.

Option 1 : just nanopore reads

sample,fastq
test,long_reads_low_depth.fastq.gz

Option 2 : nanopore reads and at least one sample has Illumina paired-end fastq files

sample,fastq,fastq_1,fastq_2
sample1,sample1.fastq.gz,sample1_R1.fastq.gz,sample1_R2.fastq.gz
sample2,sample2.fastq.gz,,

Switching assemblers

There are currently several options for assembly

flye (default)
raven
unicycler (requires short reads for hybrid assembly)

These are specified with the assembler paramater. If Illumina reads are found, then flye and raven assemblies will be polished with those reads.

Note: more than one assembler can be chosen (i.e. params.assembler = 'flye,raven'). This will run the input files on each assembler listed. Listing an assembler more than once will not create additional assemblies with that tool (i.e. params.assembler = 'flye,flye,flye' will still only run the input files through flye once).

Reading the sequencing summary file

Although not used for anything else, the sequencing summary file can be read in and put through nanoplot to visualize the quality of a sequencing run. This is an optional file and can be set with 'params.sequencing_summary'.

nextflow run UPHL-BioNGS/Donut_Falls -profile singularity --sequencing_summary <sequencing summary file>

WARNING : Does not work with older versions of the summary file.

Examples

# nanopore assembly with flye followed by polishing if illumina files are supplied
nextflow run UPHL-BioNGS/Donut_Falls -profile singularity --sample_sheet sample_sheet.csv

# or with docker and specifying the assembler
nextflow run UPHL-BioNGS/Donut_Falls -profile singularity --sample_sheet sample_sheet.csv --assembler flye

# hybrid assembly with unicycler where both nanopore and illumina files are required
nextflow run UPHL-BioNGS/Donut_Falls -profile singularity --sample_sheet sample_sheet.csv --assembler unicycler

# assembling with all three asssemblers
# specifying the results to be stored in 'donut_falls_test_results' instead of 'donut_falls'
# using docker instead of singularity
nextflow run UPHL-BioNGS/Donut_Falls -profile docker --sample_sheet sample_sheet.csv --assembler unicycler,flye,raven


# using some test files (requires internet connection)
nextflow run UPHL-BioNGS/Donut_Falls -profile docker --sample_sheet sample_sheet.csv --test

# same as above
nextflow run UPHL-BioNGS/Donut_Falls -profile docker,test --sample_sheet sample_sheet.csv

Credits

Donut Falls would not be possible without

bandage : visualize gfa files
busco : assessment of assembly quality
bwa : aligning reads for polypolish
circulocov : read depth per contig
dnaapler : rotation
fastp : cleaning illumina reads (default values) and nanopore reads (minimum length = 1,000 & minimum Q = 12)
flye : de novo assembly (default assembler)
gfastats : assessment of assembly
medaka : polishing with nanopore reads
multiqc : amalgamation of results
nanoplot : fastq file QC visualization
polypolish : reduces sequencing artefacts through polishing with Illumina reads
pypolca : reduces sequencing artefacts through polishing with Illumina reads
rasusa : subsampling nanopore reads to 150X depth
raven : de novo assembly option (params.assembler = 'raven')
unicycler : hybrid assembly option (params.assembler = 'unicycler')