NanoVar-Micro

Nanopore Microbial Variant Calling Pipeline

This pipeline runs dorado for high accuracy basecalling and alignment to reference genomes creating a BAM file that can be used as an input the bwa_pipeline_nanopore. This pipeline makes variant calls by using samtools/bcftools, varscan and gatk. It annotates the variants by using snpEff/snpSift and combines/collates variants that can be filtered based on the number of callers.

Setup of the environment

Basecalling with dorado requires GPU. Basecalling/alignment steps were performed in Macboo Pro (M1),3.2 Ghz, 10 CPU/16 GPU, 32GB Memory.

It took about ~13 hours to make basecalling and alignment to merged Dvh and Mmp genomes by using high quality basecalling model.

Setup directory structure

• Nanopore

  • code

  • data

    • EPD9
      • barcode07
        • Run1, Run2, Run3, Run5

  • output

    • EPD9
      • barcode07
        • alignment_results
        • samtools_results
        • gatk_results
        • varscan_results
        • combined_output
          • dvh
          • mmp
        • snpeff_results
          • dvh
          • mmp
      • barcode07
        • basecalls

  • model

  • reference

  • dorado

Install dorado

Download provided binaries for the relevant platform from dorado Github repository.

Unpack the dorado under the dorado directory

Download the relevant dorado model

Download the relevant model from dorado gihub repository. We have used [email protected] model for the latest high accuracy basecalling.

code/dorado-0.4.3-osx-arm64/bin/dorado download --model [email protected]

model file is placed under the model directory

Setting up POD5 for merging and converting fast5 files

pip install pod5

Convert .fast5 files from all the runs into one final .pod5 file

pod5 convert fast5 -r Run1/barcode07/*.fast5 Run2/barcode07/*.fast5 Run3/barcode07/*.fast5 Run4/barcode07/*.fast5 --output barcode07_combined.pod5

Run dorado

dorado/dorado-0.4.3-osx-arm64/bin/dorado basecaller model/[email protected] data/EPD9/barcode07/barcode07_combined.pod5 --reference reference/DvH_Mmp_merged_dna.genome.fasta > output/EPD9/barcode07/basecalls/barcode07.bam

Running trio_variant_calling pipeline

Trio Variant Calling Pipeline uses BAM input file from dorado and calls variant by using samtools, gatk and varscan. Annotations are performed with snpEFF and variants are collated.

• Requirements
  • samtools
  • bcftools
  • varscan
  • gatk
  • snpEff