massgenotyping

Python package for microsatellite genotyping from highly multiplexed amplicon sequencing data

Features

Semi-automatic genotyping optimized for amplicon sequencing data of microsatellite loci
Visual genotyping with interactive plots
Fast SSR search in sequences
Automatic grouping and naming of alleles based on polymorphisms in both SSR and non-SSR regions
Support for multi-core processing

Requirements

Python 3.8 or higher
NGmerge
MAFFT
BLASTn (included in BLAST+ command line applications provided by NCBI)
Optional: ripgrep

Installation

Install from PyPI

pip install massgenotyping

Install the latest version from a Git repository

pip install git+https://github.com/kohyamat/massgenotyping

Usage

mgt [-h] SUBCOMMAND [OPTIONS]

Subcommand list:

demultiplex: demultiplex raw amplicon sequences based on primer sequences
merge-pairs: merge paired-end reads
denoise: reduce any noise that may have been generated during sequencing and PCR
filter: filtering for erroneous sequence variants and screening for putative alleles
allele-check: check allele candidates and create an allele database
allele-call: assign alleles to raw amplicon sequences
show-alignment: show a sequence alingment

The details of the options for each subcommand can be checked by mgt SUBCOMMAND -h.

Tutorials with example data

Here's a step-by-step tutorial using the example data.

1. Demultiplex raw amplicon sequences based on primer sequences

As a first step, the sequence data is split based on the primer sequence. The input can be one or two sequence files in the FASTQ format, or a directory containing multiple sequence files. Primer sequences can be read from CSV or FASTA files. Please check the example data for the format of the input data.

mgt demultiplex examples/sequence_data -g "*_R[12]_*" -m examples/marker_data.csv

The result files are written in subdirectories within the output directory (./project by default) for each marker.

2. Merge paired-end reads and trim primer sequecnes

For the paired-end sequencing data, the respective sequence pairs are merged using NGmerge program. The following command removes the the primer sequences after merging sequence pairs.

mgt merge-pairs ./project -m examples/marker_data.csv --trim-primer

For single-end data, this step can be skipped. The removal of the primer sequence can also be performed in the step 1.

3. Reduce noise (optional but recommended)

This step corrects any noise (very low-frequency point mutations) that may have been generated during sequencing or PCR. This step is not necessarily required, but it will make the following step easier.

mgt denoise ./project/*/*_merged.fastq.gz

4. Filter out erroneous sequence variants

In this step, the sequence of putative alleles is extracted for each marker in each sample, while removing any erroneous sequence variants, such as 'stutter' sequences. After some rough filtering, an interactive plot allows you to choose which sequence variants to keep. You can skip this visual-checking procedure with the --force-no-visual-check option.

mgt filter ./project -m examples/marker_data.csv

5. Check a multiple sequence alignment and make an allele database

The database is created after checking the alignment of the putative allele sequences. If necessary, you can further filter out the erroneous sequence variants.

mgt allele-check ./project

6. Assign alleles to raw amplicon sequences

Finally, the following command perform a BLASTn search against the database created for each marker and assign alleles to the raw sequence data. The genotype tables are created within the output directory.

mgt allele-call ./project -m examples/marker_data.csv

Screenshots

Figure 1. Checking the multiple sequence alignment across the samples (STEP 5).

Figure 2. Visual genotyping (STEP 6).

Contributing to massgenotyping

Contributions of any kind are welcome!

License

MIT