RAMIFI

Recombinant And Mixed-Infection Finder for SARS-CoV-2 sample. It takes input from aligned bam file (aligned to NC_045512) based on defined mutation list json file provided in the repo and output recombinant and parents reads in .bam and .tsv file with associated stats file.

Design Diagram

Dependencies

Programming/Scripting languages

• Python >=v3.8
  • The pipeline has been tested in v3.8.10

Python packages

• pandas >=1.2.4
• pysam >= 0.16.0.1
• importlib-resources>=5.7.1

Optional packages

• plotly >=4.7.1
• kaleido >= 0.2.1
• biopython >= 1.78

Installation

Install by pip

pip install ramifi

Install from source

Clone the ramifi repository.

git clone https://github.com/LANL-Bioinformatics/ramifi

Then change directory to ramifi and install.

cd ramifi
pip install .

If the installation was succesful, you should be able to type ramifi -h and get a help message on how to use the tool.

ramifi -h

Usage

usage: ramifi.py [-h] [--refacc [STR]] [--minMixAF [FLOAT]] [--maxMixAF [FLOAT]] [--minMixed_n [INT]] [--minReadCount [INT]]
                 [--lineageMutation [FILE]] [--variantMutation [FILE]] [--mutations_af_plot] [--verbose] [--version] --bam [FILE]
                 [--vcf [File]] [--tsv [FILE]] [--outbam [File]] [-eo [PATH]] [--igv [PATH]] [--igv_variants]

Script to do recombinant read analysis

optional arguments:
  -h, --help            show this help message and exit
  --refacc [STR]        reference accession used in bam [default: NC_045512.2]
  --minMixAF [FLOAT]    minimum alleic frequency for checking mixed mutations on vcf [default:0.2]
  --maxMixAF [FLOAT]    maximum alleic frequency for checking mixed mutations on vcf [default:0.8]
  --minMixed_n [INT]    threshold of mixed mutations count for vcf.
  --minReadCount [INT]  threshold of read with variant count when no vcf provided.
  --lineageMutation [FILE]
                        lineage mutation json file [default: variant_mutation.json]
  --variantMutation [FILE]
                        variant mutation json file [default: lineage_mutation.json]
  --mutations_af_plot   generate mutations_af_plot (when --vcf provided)
  --verbose             Show more infomration in log
  --version             show program's version number and exit

Input:
  --bam [FILE]          <Required> bam file
  --vcf [File]          <Optional> vcf file which will infer the two parents of recombinant_variants

Output:
  --tsv [FILE]          output file name [default: recombinant_reads.tsv]
  --outbam [File]       output recombinant reads in bam file [default: recombinant_reads.bam]

EDGE COVID-19 Options:
  options specific used for EDGE COVID-19

  -eo [PATH], --ec19_projdir [PATH]
                        ec-19 project directory
  --igv [PATH]          igv.html relative path
  --igv_variants        add variants igv track

Test

cd tests
./runTest.sh

Outputs

-- recombinant_reads.stats: counts

total	mapped	unmapped	mutation_reads	parents	recomb1_reads	recomb2_reads	recombx_reads	parent1_reads	parent2_reads	recomb1_perc	recomb2_perc	recombx_perc
64355	64355	0	5203	Omicron,Delta	162	175	18	489	730	10.29	11.11	1.14

-- recombinant_reads.tsv

read_name	start	end	mutaions_json	note
HMVN7DRXY:2:2153:21802:16078	21566	21859	{21618: ['Delta'], 21846: ['Iota', 'Mu', 'Omicron']}	recombinant 2
HMVN7DRXY:2:2166:28574:36229	21732	21883	{21762: ['Eta', 'Omicron'], 21846: ['Iota', 'Mu', 'Omicron']}	parent Omicron
HMVN7DRXY:2:2215:29749:15217	22867	22994	{22917: ['Delta', 'Epsilon', 'Kappa'], 22992: ['rev of Omicron']}	parent Delta
HMVN7DRXY:2:2105:30572:25160	22865	23023	{22917: ['rev of Delta Epsilon Kappa'], 22992: ['rev of Omicron'], 22995: ['Delta', 'Omicron'], 23013: ['rev of Omicron']}	recombinant 1
HMVN7DRXY:2:2127:18304:18850	24058	24518	{24130: ['Omicron'], 24469: ['rev of Omicron'], 24503: ['Omicron']}	recombinant x
etc ...

-- recombinant_reads_by_cross_region.tsv

Cross_region	Reads
11201-11283	{"recomb1": ["HMVN7DRXY:2:2150:13015:23750", "HMVN7DRXY:2:2124:23746:28776", "HMVN7DRXY:2:2232:6216:33395"], "recomb2": ["HMVN7DRXY:2:2122:27624:23062"]}
11283-11537	{"recomb2": ["HMVN7DRXY:2:2126:12825:30154", "HMVN7DRXY:2:2126:15302:29121"]}
21618-21846	{"recomb2": ["HMVN7DRXY:2:2153:21802:16078", "HMVN7DRXY:2:2105:22996:5682"]}
etc ...

-- recombinant_reads.parent1.bam

-- recombinant_reads.parent1.bam.bai

-- recombinant_reads.parent2.bam

-- recombinant_reads.parent2.bam.bai

-- recombinant_reads.recomb1.bam

-- recombinant_reads.recomb1.bam.bai

-- recombinant_reads.recomb2.bam

-- recombinant_reads.recomb2.bam.bai

-- recombinant_reads.recombx.bam

-- recombinant_reads.recombx.bam.bai

-- recombinant_reads.mutations_af_plot.html

-- recombinant_reads.mutations_af_plot_genomeview.html

Data visualization

The recombinant_reads.bam, ramifi/data/variants_mutation.gff and ramifi/data/NC_045512.fasta can be loaded into IGV.

Example: IGV Link: https://chienchi.github.io/ramifi/igv-webapp

Custom mutation list

User can custom mustaion list formated as same defined mutation list json file provided in the repo to check other variant/lineage co-infection/recombinant. When run ramifi, the custom mutation list will be taken in by the option flag --variantMutation.

For example:

{
    "Alpha": {
        "A:23063:T": "S:N501Y",
        "A:23403:G": "S:D614G",
        ...
        "del:21991:3": "S:Y144*"
        ...
    },
    "Beta": {
        "A:10323:G": "ORF1a:K3353R",
        "A:21801:C": "S:D80A",
        "A:22206:G": "S:D215G",
        "A:23063:T": "S:N501Y"
        ...
    },
    "BA.2": {
        ...
    }
}

NCBI TRACE Lineage Definitions Weekly Update Site: https://ftp.ncbi.nlm.nih.gov/pub/ACTIV-TRACE/

Remove package:

pip uninstall ramifi

Citing RAMIFI

This work is currently unpublished. If you are making use of this package, we would appreciate if you gave credit to our repository.

License

RAMIFI is distributed as open-source software under GPLv3 LICENSE and the license file included in the RAMIFI distribution.

LANL open source approval reference C22090.

© 2023. Triad National Security, LLC. All rights reserved. This program was produced under U.S. Government contract 89233218CNA000001 for Los Alamos National Laboratory (LANL), which is operated by Triad National Security, LLC for the U.S. Department of Energy/National Nuclear Security Administration. All rights in the program are reserved by Triad National Security, LLC, and the U.S. Department of Energy/National Nuclear Security Administration. The Government is granted for itself and others acting on its behalf a nonexclusive, paid-up, irrevocable worldwide license in this material to reproduce, prepare derivative works, distribute copies to the public, perform publicly and display publicly, and to permit others to do so.