CECRET

A workflow for generating consensus sequences from single or paired-end fastq.gz or fastq reads from amplicon prepared Illumina libraries.

USAGE

usage: staphb-wf [optional arguments] <workflow> [workflow arguments] cecret [--reads_type {paired,single}] [--output <output_path>] [--profile {docker,singularity}] [--config CONFIG] [--get_config] [--resume] [reads_path]

Default Usage

staphb-wf cecret Sequencing_reads

Getting a config file for User-supplied goals

staphb-wf cecret --get_config

Using single-end instead of paired-end reads

staphb-wf cecret --reads_type single Sequencing_reads

Cecret can actually handle both read types at the same time, as long as both types are reads are in separate directories. Specify the single end reads in the config file params.single_reads = <directry with single reads> and set the reads_path to the directory with paired end reads.

Annotating a collection of fastas

staphb-wf cecret --annotation fastas

Note: set params.relatedness = true in order to get a multiple sequence alignment, SNP matrix, and newick file for the collection of fastas.

Required parameters :

• primer_bed bedfile for primer sequences
  • Default is artic’s SARS-CoV-2 V3 primer set
  • Change to user-supplied bedfile with params.primer_bed
• reference_genome fasta file of genome to align to
  • Default is MN908947.3/SARS-CoV-2
  • Change to user-supplied fasta file with params.reference_genome

Optional (recommended) parameters :

• gff_file for ivar variants (recommended)
  • Default is MN908947.3/SARS-CoV-2
  • Change to user-supplied gff file with params.gff_file
  • If not using a gff file, set params.ivar_variants = false
• kraken2 (recommended)
  • Default is false
  • Set params.kraken2 = true and kraken2_db = <path to kraken2 database>
• amplicon file
  • Default is the amplicons from artic’s V3 primers.
  • Change to user-supplied bedfile with params.amplicon_bed
  • If not using, set params.bedtools_multicov = false
• Creating a multiple sequencing alignment, SNP matrix, and treefile with mafft, snp-dist, and iqtree
  • Default is false
  • If this is desired, set params.relatedness = true

This workflow is also available as a standalone repository, https://github.com/UPHL-BioNGS/Cecret, with extended documentation.

Questions Worth Asking

How is cecret different than monroe?

It’s not all that different. monroe uses minimap2 for mapping/aligning and cleans reads with bbduk and trimmomatic. Running the aligned reads through ivar for primer trimming and consensus creation is the core for both workflows.

What if I am using an amplicon based library that is not SARS-CoV-2?

Change the following relevant paramters:

• params.reference_genome
• params.primer_bed
• params.amplicon_bed or params.bedtools_multicov = false
• params.gff_file or params.ivar_variants = false
• params.pangolin = false
• params.nextclade = false
• params.vadr = false or create a new vadr container with the appropriate build and adjust the parameters of the vadr process in a config file
• params.kraken2_organism = "<organism name>" or keep params.kraken2 = false

How can I tell if certain amplicons are failing?

There are two ways to do this.

With bedtools multicov :

cecret/bedtools_multicov has a file for each sample. This is standard bedtools multicov output, so it doesn’t have a header.

• Column 1 : The reference
• Column 2 : Start of amplicon
• Column 3 : End of amplicon
• Column 4 : Amplicon number
• Column 5-6 : version number and strand from bedfile
• Column 7 : (Column G) is the depth observed for that amplicon for that sample.

With samtools ampliconstats :

cecret/samtools_ampliconstats has a file for each sample Row number 126 (FDEPTH) has a column for each amplicon (also without a header). To get this row for all of your samples, you can grep the keyword “FDEPTH” from each sample.

grep "^FDEPTH" cecret/samtools_ampliconstats/* > samtools_ampliconstats_all.tsv

This workflow has too many bells and whistles. I really only care about generating a consensus fasta. How do I get rid of all the extras?

Change the parameters in your config file and set most of them to false.

params.fastqc = false
params.ivar_variants = false
params.samtools_stats = false
params.samtools_coverage = false
params.samtools_flagstat = false
params.samtools_depth = false
params.bedtools_multicov = false
params.samtools_ampliconstats = false
params.samtools_plot_ampliconstats = false
params.pangolin = false
params.nextclade = false
params.vadr = false

And, yes, this means I added some bells and whistles so you could turn off the bells and whistles. /irony

Where do I find the rest of the documentation?

Cecret’s standalone workflow repository : https://github.com/UPHL-BioNGS/Cecret