sn_assembleAll_reference.pl.md

SYNOPSIS

sn_assembleAll_reference.pl.md

Runs a reference assembly against a reference genome assembly and produces assembly metrics.

Software requirements

• Bowtie2
• samtools
• bcftools
• trimmomatic
• seqtk

Algorithm

Database building

• Uses reference_fasta_id to download a fasta and genbank file from NCBI
  • Found in taxonProperties.conf
  • Can use comma separated list, e.g., reference_fasta_id=MH185784,MH185777,MH185772,MN367319,MN367321,MN367323,MN367326,MH430075
• Downloads to SneakerNet installation, into a subfolder db/fasta
• Indexes the fasta file with samtools and bowtie2-build
• Gene annotations are not indexed and can be read on the fly

Assembly

• Downloads the reference genome into your installation directory
• Trims adapters with trimmomatic
  • primers are found in taxonProperties.conf
  • e.g., primers_bed_url=https://raw.githubusercontent.com/artic-network/primer-schemes/master/nCoV-2019/V3/nCoV-2019.primer.bed
  • If no primers, then will not trim and will produce a warning
• Maps reads against genome with bowtie2
• Calls SNPs with samtools
• Filters SNPs
• Consensus fasta from SNPs and the reference genome

Annotation

• copies coordinates of reference genome genes to assembly
• Counts intact coding sequences designated by the CDS tag in the genbank file
• Counts intact coding sequences in the new assembly

Outputs

Assemblies are found in SneakerNet/assemblies/[samplename]/*.fasta

Some intermediate files are in `SneakerNet/assemblies/[samplename]/consensus

• depth.tsv - output of samtools depth
• out.vcf.gz.masked.vcf.gz and related csi file
• sorted.bam and related bai file

A graph of all depths of coverage in SneakerNet/forEmail/depth.png

Table with columns

• File - the sample name with some file extensions
• genomeLength
• N50
• longestContig
• numContigs
• avgContigLength
• assemblyScore - log(N50/numContigs * penalty) where a genome is penalized for being different than the expected genome length
• minContigLength
• expectedGenomeLength
• kmer21 - a quantifier for how much 21-mers are duplicated. A marker for overassembly.
• GC
• effectiveCoverage - the number of mapped base pairs against the assembly. Will be lower than the coverage noted in readMetrics.
• percentNs - the percentage of Ns in the assembly, from 0 to 1
• altCdsCount - number of CDS in new assembly
• refCdsCount - number of CDS in reference assembly
• expectedCdsPercentage - altCdsCount / refCdsCount
• longestContiguous - the longest contiguous part of the genome between any two Ns