MMG.sh —— a script for evolutionary and ecological studies using Illumina sequencers for mixed-species samples
Mitochondrial metagenomics (MMG) pipeline for the rapid mitogenome assembly, integrating a fast, accurate read mapper for filtering non-mitochondrial reads, a seed-and-extend assembler for assembling species-specific mitogenomes while detecting ‘noisy’ species/sequences potentially obstructing target assembly.
MMG assembly procedure for each dataset was completed in a few hours on desktop PCs while maintaining high accuracy and completeness except for some very closely related taxa. Excluding ‘noisy’ reads including chimera of non-targeted species could improve the target assembly, particularly for those closely related-species.
Some bioinformatic tools are neccessary for above scripts. Most of them are recommended to be added into the environmental paths. Softwares, versions and source ate listed below.
NextGenMap v0.5.5 (https://github.com/Cibiv/NextGenMap/)
SAMtools v1.9 (https://github.com/samtools/samtools)
NOVOPlasty v4.3.1 (https://github.com/ndierckx/NOVOPlasty)
Seqkit v2.0.0 (https://github.com/shenwei356/seqkit)
Vsearch v2.14.2 (https://github.com/torognes/vsearch)
● Merge all the forward or reverse reads：
cat *.1.fq | pigz -p $THREADS -c > 1.fq.gz
cat *.2.fq | pigz -p $THREADS -c > 2.fq.gz
● Assemble the mitochondrial genomes using MMG.sh:
Type 'bash MMG.sh'
√ Some tips:
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The script will check the installation directory of NextGenMap, NOVOPlasty, Samtools, Seqkit and Vsearch automatically. Please input the installation directory (absolute path, e.g. /usr/local/bin) as prompted if is not found.
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Input the name (with its path) of the reference mitogenome FASTA file used for filtering non-mitochondrial reads. They are usually downloaded from NCBI based on target taxa.
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Input the name (with its path) of forward Fastq file (e.g. illumina.R1.fq.gz).
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Input the name (with its path) of reverse Fastq file (e.g. illumina.R2.fq.gz).
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Input the name (with its path) of seed file, e.g. COI barcode sequences of each species.
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Input the read length of sequencing data (e.g. 150, 250).
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Input the number of threads/cores (e.g. 8).
√ Chcek these four notes after the assembly carefully although more details have been described in this script:
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Check the file '1-assembly/seeds_vs_mitogenome.txt', which aligned seed sequecnes to the assemblies. The identity (the third column) and the seed cover ratio (the fourth column) may be useful for the final determination.
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For the species failed to assemble (listed in 1-assembly/round2/list.fail), carefully scan their NOVOPlasty assembly progress in the folder 1-assembly/roundX/ and encourage to assemble them separately by adjusting parameters, such as k-mer values. Some 'failed' species may be due to their shorter length (< 10,000 bp, see the file 'list.incomplete'), and you can recover them to the 'correct' ones depending on your aims.
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Check the file '2-chimera/possible_chimera.list', which listed the possible chimera sequences. Carefully curate them! Aligning results '2-chimera/XXX/blast.out' and new assembly results in the same folder may provide some hints.
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All assembled sequences are deposited in 1-assembly/novoplasty.fasta. Don't forget to delete chimera or replace the incorrect sequences!
Du, S.; Dong, J.; Godeiro, N.N.; Wu, J.; Zhang, F. Advancing Mitochondrial Metagenomics: A New Assembly Strategy and Validating the Power of Seed-Based Approach. Diversity 2022, 14, 317. https://doi.org/10.3390/d14050317
Please send emails to Dr. Feng Zhang (xtmtd.zf@gmail.com).