Mitohelper

Citation

Jean Lim, S, Thompson, LR. Mitohelper: A mitochondrial reference sequence analysis tool for fish eDNA studies. Environmental DNA. 2021; 00: 1– 10. https://doi.org/10.1002/edn3.187

Mitohelper is a Python-based mitochondrial reference sequence analysis tool for fish eDNA studies. It is useful for researchers interested in:

• finding out whether mitochondrial reference sequences exist for specific fish species/taxonomy (getrecord command in mitohelper.py)
• finding out which specific region of a mitochondrial gene has been sequenced (by aligning with a reference sequence) (getalignment command in mitohelper.py)
• using pre-formatted QIIME-compatible ribosomal RNA (12S or 12S+16S+18S) sequence and taxonomy databases for downstream analysis (available in the QIIME-compatible folder)
• knowing more about our data processing pipeline and mitohelper algorithm (visit the DevWiki for all the nitty gritty details!)

Dependencies

• Tested on python 3.6.10
• For local blastn searches, NCBI BLAST+ executables (specifically blastn) must be installed and in the system path

Required python modules:

• click (v7.1.2)

• matplotlib (v3.3.4)

• pandas (v0.25.3)

• seaborn (v0.10.1)

  Installation:

  • pip:

    pip install -r requirements.txt
    

  • conda:

    conda install --file requirements.txt
    

Usage

mitohelper.py --help

Usage: mitohelper.py [OPTIONS] COMMAND [ARGS]...

Options:
  --help  Show this message and exit.

Commands:
  getalignment  Pairwise align input sequences against a reference
  getrecord     Retrieve fish mitochondrial records from taxa list

mitohelper.py getrecord --help

Usage: mitohelper.py getrecord [OPTIONS]

  Retrieve fish mitochondrial records from taxa list

Options:
  -i, --input_file TEXT           Input query file (e.g. input.txt)
                                  [required]

  -o, --output_prefix TEXT        Output prefix (e.g. OUT)  [required]
  -d, --database_file TEXT        Database file (e.g.
                                  mitofish.all.Mar2021.tsv)  [required]

  -l, --tax_level [1|2|3|4|5|6|7]
                                  The taxonomic level of the search (e.g 7 for
                                  species, 6 for genus etc)

  --fasta / --no-fasta            Generate FASTA file output containing
                                  sequences of all matching hits
                                  (default=FALSE)

  --taxout / --no-tax             Generate taxonomy file output for all
                                  matching hits (default=FALSE)

  --help                          Show this message and exit

Reference database files can be downloaded from

• mitofish.all.Apr2024.tsv (877,543 records)
• mitofish.12S.Apr2024.tsv (59,616 records)
• mitofish.12S.Apr2024_NR.fasta (FASTA file of 12S rRNA gene records)
• mitofish.COI.Apr2024.tsv (327,853 records)

• Usage example:

mitohelper.py getrecord -i testdata/species.query.txt -o testdata/getrecordOUT -d mitofish.all.Sep2023.tsv -l 7 --fasta --taxout

• Input file example (testdata/species.query.txt):

Abraliopsis pfefferi
Ahliesaurus berryi
Alepisaurus FEROX
anotopterus pharao
AHLIESAURUS BERRYI

• Screen output:

=== Searching query #1: <Abraliopsis pfefferi> ===
=== Searching query #2: <Ahliesaurus berryi> ===
Search string:Ahliesaurus berryi	Taxonomic level:L7	Hits:2
=== Searching query #3: <Alepisaurus FEROX> ===
Search string:Alepisaurus ferox	Taxonomic level:L7	Hits:32
=== Searching query #4: <anotopterus pharao> ===
Search string:Anotopterus pharao	Taxonomic level:L7	Hits:9
=== Searching query #5: <AHLIESAURUS BERRYI> ===
DUPLICATE WARNING: Query has already been processed!
==== Run complete! ===
Accession numbers of subspecies hits and description saved in testdata/getrecordOUT_L7_hits.tsv
FASTA-formatted sequences saved in testdata/getrecordOUT_L7.fasta
Output taxonomy file saved in testdata/getrecordOUT_L7.taxonomy.tsv

• Output TSV file columns and example (testdata/getrecordOUT_L7_hits.tsv):

1. Query
2. Accession - NCBI accession
3. Gene definition - Gene definition in GenBank record
4. taxid - NCBI taxonomy ID
5. Superkingdom
6. Phylum
7. Class
8. Order
9. Family
10. Genus
11. Species
12. Sequence
13. OrderID - Order classification in "Fishes of the World, 5th edition" (Nelson et al. 2016)
14. FamilyID - Family classification in "Fishes of the World, 5th edition" (Nelson et al. 2016)

Query   Accession       Gene definition taxid   Superkingdom    Phylum  Class   Order   Family  Genus   Species Sequence        OrderID FamilyID
Ahliesaurus berryi      EU366544        Ahliesaurus berryi voucher MCZ 161662 cytochrome oxidase subunit I (COI) gene, partial cds; mitochondrial       509771  Eukaryota       Chordata        Actinopteri     Aulopiformes    Notosudidae     Ahliesaurus     Ahliesaurus berryi      GTGAACATGAGGTGGGCTCAGACGATAAAGCCTAGGAGGCCGATTGCTATCATAGCTCAGACCATGCCCATGTAGCCAAAGGGTTCTTTTTTCCCTGAATAGTAGGCTACGATGTGGGAGATCATACCAAAGCCGGGGAGAATAAGAATGTAGACCTCTGGGTGACCAAAGAATCAGAACAGGTGCTGGTAAAGGATGGGGTCTCCGCCCCCTGCCGGGTCAAAGAAGGTGGTGTTCAGGTTTCGGTCAGTTAGAAGTATTGTAATGCCTGCCGCTAGAACGGGGAGGGAGAGTAAAAGAAGGACGGCAGTAATAAGGACTGCTCAGACGAAGAGGGGAGTTTGGTACTGGGTGATGGCGGGGGGTTTTATGTTAATAATTGTTGTGATGAAGTTAATGGCACCCAGGATAGAGGAGATACCTGCCAGGTGGAGGGAGAAGATGGTTAGGTCTACGGAAGCTCCTGCATGGGCCAGGTTGCTGGCGAGAGGCGGATACACAGTTCATCCTGTTCCGGCCCCGGCTTCTACAGCGGAGGAGGCTAGGAGTAGAAGGAAGGATGGGGGGAGTAGTCAAAAGCTCATGTTGTTCATTCGGGGGAATGCCATGTCAGGCGCCCCGATCATAAGAGGGATAAGTCAGTTTCCGAACCCACCGATCATAATTGGTATTACCATGAAAAAAATTATTACGAAAGCGTGGGCAGTAACGATAACATTGTAAATCTGGTCGTCTCCTAAAAGGGCTCCGGGTTGGCTTAGCTCAGCTCGGATGAGAAGGCT  44      214
Ahliesaurus berryi      KF929574        Ahliesaurus berryi voucher KUT 5285 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial 509771  Eukaryota       Chordata        Actinopteri     Aulopiformes    Notosudidae     Ahliesaurus     Ahliesaurus berryi      CCTCTACCTACTATTTGGTGCCTGGGCCGGGATGGTGGGTACAGCCCTAAGCCTTCTCATCCGAGCTGAGCTAAGCCAACCCGGAGCCCTTTTAGGAGACGACCAGATTTACAATGTTATCGTTACTGCCCACGCTTTCGTAATAATTTTTTTCATGGTAATACCAATTATGATCGGTGGGTTCGGAAACTGACTTATCCCTCTTATGATCGGGGCGCCTGACATGGCATTCCCCCGAATGAACAACATGAGCTTTTGACTACTCCCCCCATCCTTCCTTCTACTCCTAGCCTCCTCCGCTGTAGAAGCCGGGGCCGGAACAGGATGAACTGTGTATCCGCCTCTCGCCAGCAACCTGGCCCATGCAGGAGCTTCCGTAGACCTAACCATCTTCTCCCTCCACCTGGCAGGTATCTCCTCTATCCTGGGTGCCATTAACTTCATCACAACAATTATTAACATAAAACCCCCCGCCATCACCCAGTACCAAACTCCCCTCTTCGTCTGAGCAGTCCTTATTACTGCCGTCCTTCTTTTACTCTCCCTCCCCGTTCTAGCGGCAGGCATTACAATACTTCTAACTGACCGAAACCTGAACACCACCTTCTTTGACCCGGCAGGGGGCGGAGACCCCATCCTTTACCAGCACCTG    44      214

• Output taxonomy file example (testdata/getrecordOUT_L7_taxonomy.tsv):

The output taxonomy file can be directly imported into QIIME 2

Feature ID      Taxon
EU366544        d__Eukaryota; p__Chordata; c__Actinopteri; o__Aulopiformes; f__Notosudidae; g__Ahliesaurus; s__Ahliesaurus berryi
KF929574        d__Eukaryota; p__Chordata; c__Actinopteri; o__Aulopiformes; f__Notosudidae; g__Ahliesaurus; s__Ahliesaurus berryi
AF092181        d__Eukaryota; p__Chordata; c__Actinopteri; o__Aulopiformes; f__Alepisauridae; g__Alepisaurus; s__Alepisaurus ferox
AP004211        d__Eukaryota; p__Chordata; c__Actinopteri; o__Aulopiformes; f__Alepisauridae; g__Alepisaurus; s__Alepisaurus ferox
EU366542        d__Eukaryota; p__Chordata; c__Actinopteri; o__Aulopiformes; f__Alepisauridae; g__Alepisaurus; s__Alepisaurus ferox

getalignment

mitohelper.py getalignment --help

Usage: mitohelper.py getalignment [OPTIONS]

  Pairwise align input sequences against a reference

Options:
  -i, --input_file TEXT           Input file: either blastn output file or
                                  FASTA file  [required]
  -o, --output_prefix TEXT        Output prefix (e.g. OUT)  [required]
  -r, --reference_sequence TEXT   FASTA file of a single reference sequence
                                  for blastn searches. Required for --blast
                                  option.
  --blastn-task [blastn|blastn-short|megablast|dc-megablast|none]
                                  Choice of blastn task for local similarity
                                  searches used to extract alignment positions
                                  [default:blastn-short]  [required]
  --help                          Show this message and exit.

• Usage example:

mitohelper.py getalignment -i testdata/12S.test.fasta -o testdata/blastnALN -r testdata/Zebrafish.12S.ref.fasta --blastn-task blastn

• Screen output:

==== Run complete! ===
blastn output saved in blastnALN.blastn.txt
Table of alignment positions saved in blastnALN.alnpositions.tsv
Plot of alignment positions saved in blastnALN.alnpositions.pdf

• blastn -outfmt 7 input/output (testdata/blastnALN.blastn.txt):

# BLASTN 2.6.0+
# Query: NC_002333.2:1020-1971 Danio rerio mitochondrion, complete genome
# Database: User specified sequence set (Input: Zebrafish.12S.ref.fasta)
# Fields: query acc.ver, subject acc.ver, % identity, alignment length, mismatches, gap opens, q. start, q. end, s. start, s. end, evalue, bit score
# 1 hits found
NC_002333.2:1020-1971   NC_002333.2:1020-1971   100.000 952     0       0       1       952     1       952     0.0     1718

• Graphical output with reference sequence on top (testdata/blastnALN.alnpositions.pdf):

• Tab-separated output (testdata/blastnALN.alnpositions.tsv):

Reference sequence will always be on top.

Accession       Start   End     Bit_Score
NC_002333.2:1020-1971   1       952     1718
AB938103        249     348     37
AB006953        1       945     1099
AB015962        102     945     484
AB016274        17      945     549
AB018224        2       825     473
AB018225        2       804     471
AB018226        1       825     491
AB018227        1       804     520
AB018228        1       825     504
AB018229        1       804     506
AB018230        1       825     513