Pipeline Overview

Pipeline Summary:

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QC

1. PhiX174 read removal and adapter removal using BBDuK
2. Filtering, trimming, and base correction using fastp that includes:
   • quality trimming with a window size of 20 and quality of 30
   • quality pruning at 3' and 5' ends
   • removal of short reads
   • forced polyG tail trimming
3. Contamination check of trimmed reads using Kraken2 and the MiniKraken2_v2_8GB.

Analysis of Trimmed Reads

4. QC Metrics Generated (all data generated for paired and unpaired reads generated post-trimming):
   • Number of total reads/bases
   • Percent of reads/bases remaining (from raw sequences)
   • Number of Q20/Q30 bases
   • Percent Q20/Q30 bases

Analysis using Trimmed Reads

5. Gene detection and allele calling for antibiotic resistance (AR) srst2 in gene mode. We have curated an AR gene database that is a combination of three AR gene databases with redundancies removed and gene names standardized.

• The curated AR gene database includes complete genes from these AR gene databases:
  • AMRFinderPlus
  • ARG-ANNOT
  • ResFinder

Assembly

6. Assembly of trimmed reads using SPAdes
7. Filter reads to remove any scaffolds less than 500bp in length.

QC of Assembled Scaffolds >= 500bps

8. Assess assembly quality using QUAST and custom scripts
9. QC Metrics Generated:
   • Raw coverage (total raw bases / assembly length)
   • Trimmed coverage (total trimmed bases / assembly length)
   • Assembly ratio (assembly size / median genome size of species)

Analysis of Assembled Scaffolds >= 500bps

10. Assess genome assembly for completeness using BUSCO
11. A mash sketch is created with Mash and the .msh file is passed into FastANI for increased speed in species ID.
12. Calculate the average nucleotide identity (between genomes) using FastANI to determine species.
13. Type multiple loci to characterize isolates of microbial species using MLST
14. AR genes, hypervirulence genes and plasmid markers are detected using GAMMA. We have curated an AR gene database that is a combination of three AR gene databases with redundancies removed and gene names standardized.

• The curated database includes genes from these AR gene databases:

  • AMRFinderPlus
  • ARG-ANNOT
  • ResFinder

• Additional databases are used:

  • Database of hypervirulence genes from Russo et al.
  • PlasmidFinder

15. In addition to running Kraken2 on the trimmed reads, KRAKEN2 is run on the assembled reads using the same MiniKraken2_v2_8GB database. This additional step allows us to check if any contamination made it into the assembly.