q2-pinocchio: PaIrwise alignment of long-read NucleOtide sequence data for Classification and quality Control in HIgh-thrOughput

QIIME 2 Plugin for quality control and taxonomic classification of long sequences

Installation

Step 1: Create q2-pinocchio environment

mamba create -n q2-pinocchio -c conda-forge -c bioconda -c https://packages.qiime2.org/qiime2/2024.10/metagenome/passed/ -c defaults q2cli q2-types q2-feature-classifier minimap2 bs4 samtools gzip chopper nanoplot

Step 2: Activate q2-pinocchio environment

conda activate q2-pinocchio

Step 3: Installing python package

pip install .

Provided Actions

1. build-index

   Build a Minimap2 index database from reference sequences.

2. minimap2-search

   Search for top hits in a reference database using alignment between the query sequences and reference database sequences using Minimap2. Returns a report of the top M hits for each query (where M=maxaccepts).

3. filter-reads

   This method aligns long-read sequencing data (from a FASTQ file) to a set of reference sequences, identifying sequences that match or do not match the reference within a specified identity percentage. The alignment is performed using Minimap2, and the results are processed using Samtools.

4. extract-reads

   This method aligns long-read sequencing data (from a FASTA file) to a set of reference sequences, identifying sequences that match or do not match the reference within a specified identity percentage. The alignment is performed using Minimap2, and the results are processed using Samtools.

5. classify-consensus-minimap2

   Assign taxonomy to query sequences using Minimap2. Performs alignment between query and reference reads, then assigns consensus taxonomy to each query sequence.

6. trim

   Trim long demultiplexed sequences using Chopper tool.

7. stats

   Quality control statistics of long-read sequencing data using NanoPlot.

Examples

Download the input datasets

• build-index
  • Build Minimap2 index database

  qiime pinocchio build-index --i-reference reference.qza --o-index index.qza --verbose

• minimap2-search

  • Generate both hits and no hits for each query. Keep a maximum of one hit per query (primary).

  qiime pinocchio minimap2-search --i-query fasta_reads.qza --i-index index.qza --o-search-results paf.qza --verbose

  • Generate only hits for each query. Keep a maximum of one hit per query (primary mappings).

  qiime pinocchio minimap2-search --i-query fasta_reads.qza --i-index index.qza --o-search-results paf_only_hits.qza --p-output-no-hits false --verbose

  • Generate only hits for each query, limiting the number of hits to a maximum of 3 per query. Ensure that each hit has a minimum similarity percentage of 90% to be considered valid.

  qiime pinocchio minimap2-search --i-query fasta_reads.qza --i-index index.qza --o-search-results paf_only_hits_ma3.qza --p-maxaccepts 3 --p-output-no-hits false --verbose

• filter-reads

  • Keep mapped (single-end reads)

  qiime pinocchio filter-reads --i-query single-end-reads.qza --i-index index.qza --o-filtered-query mapped_se.qza --verbose

  • Keep unmapped (single-end reads)

  qiime pinocchio filter-reads --i-query single-end-reads.qza --i-index index.qza --p-keep unmapped --o-filtered-query unmapped_se.qza --verbose

  • Keep mapped (paired-end reads)

  qiime pinocchio filter-reads --i-query paired-end-reads.qza --i-index index.qza --o-filtered-query mapped_pe.qza --verbose

  • Keep mapped reads with mapping percentage >= 98% (paired-end reads)

  qiime pinocchio filter-reads --i-query paired-end-reads.qza --i-index index.qza --p-min-per-identity 0.98  --o-filtered-query mapped_pe_over_98p_id.qza --verbose

• extract-reads
  • Extract mapped

  qiime pinocchio extract-reads --i-sequences fasta_reads.qza --i-index index.qza --o-extracted-reads mapped_fasta.qza --verbose

  • Extract unmapped

  qiime pinocchio extract-reads --i-sequences fasta_reads.qza --i-index index.qza --p-extract unmapped --o-extracted-reads unmapped_fasta.qza --verbose

  • Extract mapped reads with mapping percentage >= 87%

  qiime pinocchio extract-reads --i-sequences fasta_reads.qza --i-index index.qza --p-min-per-identity 0.87 --o-extracted-reads mapped_fasta_ido_ver_87.qza --verbose

• classify-consensus-minimap2
  • Assign taxonomy to query sequences using Minimap2

  qiime pinocchio classify-consensus-minimap2 --i-query n1K_initial_reads_SILVA132.fna.qza --i-index ccm_index.qza --i-reference-taxonomy raw_taxonomy.qza --p-n-threads 8 --output-dir classification_output --verbose

• trim
  • Filter based on the quality (min)

  qiime pinocchio trim --i-query single-end-reads.qza --p-min-quality 7 --o-filtered-query filt_qual_min.qza --verbose

  • Filter based on the quality (max)

  qiime pinocchio trim --i-query single-end-reads.qza --p-max-quality 7 --o-filtered-query filt_qual_max.qza --verbose

  • Headcrop of all sequences ()

  qiime pinocchio trim --i-query single-end-reads.qza --p-headcrop 10 --o-filtered-query headcrop.qza --verbose

  • Filter based on the length of the sequences (min)

  qiime pinocchio trim --i-query single-end-reads.qza --p-min-length 3000 --o-filtered-query filt_len_min.qza --verbose

• stats
  • Generate a visualization to display statistics about the sequences

  qiime pinocchio stats --i-sequences single-end-reads.qza --o-visualization stats.qzv

• To open:

qiime tools view stats.qzv