title	author	date	output
README.md	Victoria Blanchard	11/08/2022	pdf_document

Data should be organised into subfolders named after the isolate

Dependencies

Before you run the code, please ensure you have these installed and added to your path:

• anaconda3
• kraken2 and Kraken2-build
  • OpenMP
  • wget
  • rsync
• phyloseq

Pipeline and associated files

To just run through the entire pipeline from the downloaded Git repository: 0.V.bombi_genome_assembler.sh - Inputs: Isolate name, Path to folder containing the raw reads for that isolate - Outputs: Fasta file with assembled genome, quality reports, all intermediate files generated in the pipeline

Otherwise, the code can be run file by file following the pipeline:

1. Filter high quality reads

   1.1.initial_set-up.sh Assuming the Git repository is cloned and immediately run, set up directory structure and set isolate name

   • Input: isolate name
   • Output: report and results directories for the isolate

   1.2.filter_high_quality_reads.sh Remove Oxford Nanopore adapters, reads from the lambda phage control genome added in the Ligation Library Preparation, and all reads with a q score lower than 10.

   • Input:
   • Outputs: One fastq file containing all reads, summary plots of the raw reads in the results directory

2. Remove contaminating Bombus terrestris DNA sequences Align the reads to the genome of the largest known contaminant - Bombus terrestris, then extract unaligned reads from the resulting SAM file into FASTA and FASTQ files.

   2.1.0.FASTQ_to_BAM_using_BWA.sh

   • Inputs: Reference sequence, Reads in FASTQ, Reads in paired FASTQ or "unpaired"
   • Output: alignment results to SAM file
   • Uses subscript: 2.1.1.SAM_how_many_reads_align.pl

   2.2.SAM_unaligned_reads_to_fasta.pl

   • Input: Sam file from 2.1
   • Output: FASTA file with unaligned reads

   2.3.SAM_unaligned_reads_to_fastq.pl

   • Input: Sam file from 2.1
   • Output: FASTA file with unaligned reads

3. Generate an assembly from reads without B. terrestris DNA Run Canu to assemble the genome on the remaining reads. This will contain some contamination that will be filtered further later. Quality assess the assembly using quast.

   3.run_canu_assembler.sh

   • Input:
   • Output:

4. Isolate microsporidia DNA from the new assembly and generate a FASTA file for matched and unmatched reads respectively. Search the unmatched reads for V. bombi primer sequences.

   4.1.0.run_busco_analysis.sh Search newly assembled genome for microsporidia BUSCOs

   • Input:
   • Outputs:
     • summary plot code: 4.1.1.busco_figure_isolate.R

   4.2.0.isolate_microsporidia_sequences.sh Extract microsporidia sequences from the first assembly into a new fasta file

   • Input:
   • Output:
   • Uses subscript: 4.2.1.get_BUSCO_matched_sequences.py

   4.3.0.check_Vb_primers.sh Ensure that all sequences from the first assembly that match V. bombi-specific primers are included in the microsporidia-specific sequences FASTA file.

   • Input:
   • Output:
   • Uses subscript: 4.3.1.FASTA_search_Vb_primers.sh

5. Generate an assembly from microsporidia-specific reads Generate a new assembly and thoroughly quality test it.

   3.run_canu_assembler.sh Re-run canu script with this new input FASTA file to assemble a genome from reads we know are from the microsporidia, and therefore presumably only V. bombi DNA

   • Input:
   • Output:

   5.1.genome_quality_assessment.sh

   • Input:
   • Output:

6. Annotate assembly

   6.run_braker2.sh

   • Input:
   • Output: