tprK pipeline

This pipeline was designed to take Illumina and PacBio files straight off the sequencer to a final comparison table of all the different variable regions with their relative frequencies, as well as various pretty plots along the way.

Table of Contents

• Setup
• Input Files
• Usage
• Common Errors

Setup

1. Install nextflow.
   • Make sure you move nextflow to a directory in your PATH variable.
2. Install docker. The first time running this program will take a while, as the docker image will take some time to build, but this is a one time thing!

Input Files

Put the following things in one folder:

• All the sequence files to run analysis on
  • PacBio Q20 reads, gzipped
  • Single-end Illumina reads trimmed and run through Trimmomatic, gzipped
  • By default the pipeline expects both PacBio and Illumina files for every sample. Running the pipeline with just PacBio or just Illumina files is possible with the --pacbio and --illumina flags respectively. However, some plots require both files to be generated and these plots will not be output.
• Metadata file. This should be a .csv with three columns: SampleName, PacBio, Illumina, shown in the table below. Make sure to include absolute paths to PacBio and Illumina files!
  • This file should be placed in the same folder as your files to be analyzed.
  • There MUST be a newline character at the end of this file to be read as a valid csv. Simply hit enter in the last row to ensure there is a valid new line.
  • Ensure that there are no special characters, including hyphens! Underscores are okay.
  • If running just Illumina or just PacBio, simply leave those columns blank (but make sure to have commas as appropriate).
  • Example metadata files (for both, just Illumina, and just PacBio) are provided in the example/ folder. The general format of the metadata file should be three columns, separated by commas, as shown:

SampleName	Illumina	PacBio
This will largely be the name used for generating tables and plots.	Should be in format Ill_[sample name].fastq.gz. The Illumina file specified for the sample name. This must match exactly the name of the matching file in the folder. This should be a trimmed file run through Trimmomatic.	Should be in format PB_[sample name].fastq.gz. The PacBio file specified for the sample name. This must match exactly the name of the matching file in the folder. This should be a Q20 file.

Usage

• Example command for just Illumina files in current directory on a laptop without many CPUs: nextflow run michellejlin/tprk -r nextflow --INPUT ./ --OUTDIR output/ --ILLUMINA --METADATA metadata.csv -resume -with-docker ubuntu:18.04 -with-trace -profile laptop
• Example command for comparing PacBio and Illumina files with specified cutoffs on the cloud with a large dataset: AWS_PROFILE=covid nextflow run michellejlin/tprk -r nextflow --INPUT example/ --OUTDIR example/output/ --METADATA metadata.csv --LARGE -resume -with-docker ubuntu:18.04 -with-trace -c ~/nextflow.covid.config -profile Cloud
• Example command for just Illumina files in current directory with a specified reference sample for variable region comparisons: nextflow run michellejlin/tprk -r nextflow --INPUT ./ --OUTDIR output/ --ILLUMINA --METADATA metadata.csv -resume -with-docker ubuntu:18.04 -with-trace --LARGE --REFERENCE inoculum_S168_trim

For a list of arguments, you can also run nextflow run michellejlin/tprk -r nextflow --help .

Command	Description
--INPUT	Input folder where gzipped fastqs are located. For current directory, ./ can be used.
--OUTDIR	Output folder where .bams and consensus fastas will be piped into.
--METADATA	Path to metadata file with specific format.
--PACBIO	Specify that there are only PacBio files to be read.
--ILLUMINA	Specify that there are only Illumina files to be read.
--LARGE	Specify that this is a large dataset. Splitting of visualizations will be done.
--REFERENCE	Specify Illumina sample name (not file), to compare others to for dot-line plots. Can be used in tandem with --LARGE.
--RF_FILTER	Specify relative frequency filter. Default is 0.2.
--COUNT_FILTER	Specify count filter. Default is 5.
--ILLUMINA_FILTER	Specify whether PacBio reads should be filtered to only include files supported by Illumina reads that reach the cutoff.
-resume	nextflow will pick up where it left off if the previous command was interrupted for some reason.
-with-docker ubuntu:18.04	Runs command with Ubuntu docker.
-with-trace	Outputs a trace.txt that shows which processes end up in which work/ folders.

Common Errors

1. incomplete final line found by readTableHeader on '/Users/uwvirongs/Documents/tprk/metadata.csv' Make sure your metadata file has a new line at the end. You can do this by simply pressing enter on the last line of your file and saving.