An automated pipeline to process ITS2 sequences from honey and plants sequences from National Honey Monitoring Scheme
** Current version: 1.2 (updated 2023-08-07) **
Install mamba if you don't already have it. You ought to follow the instruction at https://github.com/conda-forge/miniforge#mambaforge or if you have CONDA then:
conda install -c conda-forge mamba -y
In your home directory, copy and paste the following (line by line):
cd ~
mamba create -n honeypi_env -y python=3.8 progressbar2 requests rdptools itsx vsearch trim-galore bbmap seqkit -c bioconda
mamba activate honeypi_env
mamba install -c conda-forge -c bioconda biopython -y
mamba install -c anaconda pandas -y
git clone https://github.com/hsgweon/honeypi.git
pip install ./honeypi
mamba deactivate
Within R, check to see if dada2 is installed:
R
library(dada2)
If not, then install them with:
if (!requireNamespace("BiocManager", quietly = TRUE)) install.packages("BiocManager")
BiocManager::install("dada2", ask = FALSE)
If it asks "Would you like to use a personal library instead?", say yes. It will ask for a location, and again just say yes. When all done, type "quit()" and say "n".
Check again to see if the packages are all in place.
That's it!
It is highly recommended that you test HONEYPI (see 4. Testing HONEYPI) before you run it on your dataset.
Since we just created a sandbox "honeypi_env" in which all tools (except R and its packages) are installed, we need to be running honeypi and processing data within the environment:
mamba activate honeypi_env
Then, go to a directory where with your rawdata directory is located, and create a readpairslist file. This file is needed to ensure all files and sample names are correctly labelled. It does some internal checks to make sure there are no human errors with samples names etc.
N.B. rawdata is your directory with paired-end sequences*
cd honeypi/testdata
honeypi_createreadpairslist -i rawdata -o readpairslist.txt
honeypi -i rawdata -o honeypi_output --amplicontype ITS2 -l readpairslist.txt
Done... simple... isn't it? Ah, one more thing - after you finished with HONEYPI, don't forget to get out of the sandbox by:
mamba deactivate
mamba activate honeypi_env
pip uninstall honeypi
mamba deactivate
mamba env remove --name honeypi_env -y
source activate honeypi_env
cd ~/honeypi/testdata
honeypi_createreadpairslist -i rawdata -o readpairslist.txt
honeypi -i rawdata -o honeypi_output --amplicontype ITS2 -l readpairslist.txt
Look inside the output directory ("honeypi_output"), and find:
- ASVs_counts.txt
- ASVs.fasta
- ASVs_taxonomy.txt
- summary.log
(Misc) Check to see if your files have:
ASVs_counts.txt
H12BB43 H12DSPINHK9
ASV_0000000001 0 2853
ASV_0000000002 0 1836
ASV_0000000003 0 889
ASV_0000000004 0 345
ASV_0000000005 0 340
ASV_0000000006 50 200
ASV_0000000007 0 218
ASV_0000000008 0 172
ASV_0000000009 100 0
ASV_0000000010 0 69
ASV_0000000011 0 62
ASV_0000000012 0 34
ASV_0000000013 0 31
ASV_0000000014 0 14
ASV_0000000015 0 10
ASV_0000000016 0 9
ASV_0000000017 0 5
ASV_0000000018 0 3
ASV_0000000019 0 3
ASV_0000000020 0 3
ASV_0000000021 0 2
*ASVs.fasta
>ASV_0000000001
ATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCCAAGCCTTCTGGCCGAGGGCACGTCTGCCTGGGTGTCACAAATCGTCGTCCCCCCCATCCTCTCGAGGATATGGGACGGAAGCTGGTCTCCCGTGTGTTACCGCACGCGGTTGGCCAAAATCCGAGCTAAGGGCGCCAGGAGCGTCTCGACATGCGGTGGTGAATTCAAGCCTCGTAATATCGTCGGTCGTTCCGGTCCAAAAGCTCTCGATGACCCAAAGTCCTCAACGCGACCCCAGGTCAGGCGGGATCACCCGCTGAGTTTAAGCATATCAATAAGCGGAGGA
>ASV_0000000002
ATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGATGCCATTAGGCTGAAGGCACGTCTGCCTGGGCGTCACGCATGGCGTCGCCCACTCACCCCGTGCCTCTGTGGGCGGAAGGTGTGTGAGCGGATATTGGCCCCCCGTTCACGTTCGTGCTCGGTCGGTCTAAAAGGAAAGTCCCCAACGACGGACATCACGGCGAGTGGTGGTTGCCAGACCGTCCCGACGCGTCGTGCATGCTGTTCTTTGTCGTTGGCCGGCTCATCGACCCCCGAGTACCGTCAGGTACTCGGTACCTCGACTGCGACCCCAGGTCAGGCGGGATCACCCGCTGAGTTTAAGCATATCAATAAGCGGAGGA
>ASV_0000000003
ATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCTGAAGCCATCAGGTTGAAGGCACGTCTGCCTGGGCGTCACGCATTGCGTCGCTCACTCACCCCGTGCATCATTGGGCGGGCAAGTGTGTGGGCGGATATTGGCCCCCCGTTCACATTTGTGCTCGGTCGGCCTAAAAAGAAGGTCCTTGATGACGGACATCACAACAAGTGGTGGTTGCTAAACCGTCGCGCCATGTTGTGCATTATACTCCGTCGTCGGTTGCCTCATTGACCCTTAAGTGCCATTGAACTTGGTACCTCAACTGCGACCCCAGGTCAGGCGGGATTACCCGCTGAGTTTAAGCATATCAATAAGCGGAGGA
>ASV_0000000004
ATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCATTAGGCCGAGGGCACGCCTGCCTGGGCGTCACACGTCGTTGCCCCCCCCCAAACCCCTCGGGAGTTGGGCGGGACGGATGATGGCCTCCCGTGTGCTCTGTCATGCGGTTGGCATAAAAACAAGTCCTCGGCGACTAACGCCACGACAATCGGTGGTTGTCAAACCTCTGTTGCCTATCGTGTGCGCGTGTCGAGCGAGGGCTCAACAAACCATGTTGCATCGATTCGTCGATGCTTTCAACGCGACCCCAGGTCAGGCGGGGTTACCCGCTGAATTTAAGCATATCAATAAGCGGAGGA
>ASV_0000000005
ATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTTTTTGAACGCAAGTTGCGCCCGAAGCCATCCGGTTGAGGGCACGCCTGCCTGGGCGTCACGCTTCGCATCGCCCCCCACCATACATACCCAACGGGTACTAATGGTGTTTGGGGCGGAGATTGGCCTCCCGCACCTCTGATGCGGTTGGCCTAAAAATGAGTCCCCTTCAGCGGACACACGACTAGTGGTGGTTGAACAGACCCTCGTCCTTATCGTGTGTCGTGAGCTGCAAGGGAAACCCTCACCAAAGACCCTATTGCATTGTTTTTTGGACAATGCTTCGACCGCGACCCCAGGTCAGGCGGGACTACCCGCTGAGTTTAAGCATATCAATAAGCGGAGGA
>ASV_0000000006
ATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGATGCCATTAGGTTGAGGGCACGTCTGCCTGGGCGTCACATATCGAAGCCTCTCGCCAATTTCCTATATTGATAGGGGTATTGTGCAGGGCGAATGTTGGCCTCCCGTGAGCTTTATTGCCTCATGGTTGGTTGAAAATCGAGACCTTGGTAGGGTGTGCCATGATAGGTGGTGGCTGTGTTACGCACGAGACCAAGTAAGTCATGTGCTGCTCTATTGAATTTAGGCCTCTTTTACCCACATGCGTTTCGAAACGCTCGTGATGAGACCTCAGGTCAGGCGGGGCTACCCGCTGAATTTAAGCATATCAATAAGCGGAGGA
>ASV_0000000007
ATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCTGATGCCATTAGGTTGAGGGCACGTCTGCCTGGGCGTCACATATCGAAGCCTCTTGCCAATTTCCTATTGATTGGTATTGTGCAAGATGATGTTGGCCTCCCGTGAGCACCATCGCCTCATGGTTGGTTGAAAATCGAGACCTTGGTAGAGTGTGCCATGATAAATGGTGCATGTGTTAAGCACGAGACCAAACAATCATGTGCTGCTCTATTGAATTTAGCCTCTTTTACCCACATGCGTGTCTAAACGCTCGTGATGAGACCTCAGGTCAGGCGGGGCTACCCGCTGAATTTAAGCATATCAATAAGCGGAGGA
>ASV_0000000008
ATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGATGCCATTAGGTTGAGGGCACGTCTGCCTGGGTGTCACATATCGAAGCCTCCTTGCCAATTTCCCTGATTATTGTGCAGGGTGGATGTTGGCCTCCCGTGAGCTCTTTCGTCTCATGGTTGGTTGAAAATTGAGACCTTGGTAGGGTGTGCCATGATAGATGGTGGTTGTGTGACCCACGAGACCAATCATGCGCTGCTCTATTGAATTTGGCCTCCTTTACCCATATGCGTTTCCAAACGCTCGTGATGAGACCTCAGGTCAGGCGGGGCTACCCGCTGAATTTAAGCATATCAATAAGCGGAGGA
>ASV_0000000009
ATGCGATACTTGGTGTGAATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCAGGGGGCATGCCTGTTTGAGCGTCATTTCCCTCTCAAACGCTTGCGTTTGGTAGTGAGCGATACTCTTTTTGTGTGTATCTCTGAGGAGTTTGCTTGAAAGTGGGAGGCCATAGGCGGAGCCTAGCTTGAGCGTGTGGTGGAGGAACTGTGCCGAGAGGTGCAGGGCCGCGCTGCAACGCCTGGCCACGAAAACGAAGTCGTATTAGGTTTTACCGACTCGGCGAAGGAAGTAGTGGACGGGGGGAAAAGAGCGGAGCTCTCTTTTTTGTTTTGTTTGTTGATGATACGACGAGCAAGAGCAGCAGAGCCTGGCTTGAGAGAATTCACAAAGTTTGACCTCAAATCAGGTAGGATTACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA
>ASV_0000000010
ATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGACGCCTTCGGGCTGAGGGCACGCCTGCCTGGGCGTCACGCATCGCGTCGTCCCCTCCCATTCCCTCACGGGTTTGGTTATGGGACGGATAATGGCTTCCCGTTAGCTCGGTTAGCCCAAAAAGGATCCCTCATCGACGGATGTCACAACCAGTGGTGGTTGAAAGATCATTGGTGCTGTTGTGCTTCACCCTGTCGCTTGCTAGGGCATCGTCATAAACTAACGGCGTGTAATGCGCCTTCGACCGCGACCCCAGGTCAGACGGGACTACCCGCTGAGTTTAAGCATATCAATAAGCGGAGGA
>ASV_0000000011
ATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCCAAGCCTTCTGGCCGAGGGCACGTCTGCCTGGGTGTCACAAATCGTCGTCCCCCCATCCTCTCGAGGATATGGGACGGAAGCTGGTCTCCCGTGTGTTACCGCACGCGGTTGGCCAAAATCCGAGCTAAGGACGTTTTGGAGCGTCTCGACATGCGGTGGTGAATTGTAACCTCGTCATATTGTCGGTCGTTCCGGTTCAAAAGCTCTTGATGACCCAAAGTCCTCAACGCGACCCCAGGTCAGGCGGGATCACCCGCTGAGTTTAAGCATATCAATAAGCGGAGGA
>ASV_0000000012
ATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTTTTTGAACGCAAGTTGCGCCCGAAGCCATCCGGCCGAGGGCACGCCTGCCTGGGCGTCACGCATCGCGTCGCCCCCACCAAATTTCCAAATCTGGTTGGGGGCGGAGATTGGCCTCCCGTACCTGTTGTGGTTGGCCTAAAAAGGAGTCCCCTTCGGTGGACACACGACTAGTGGTGGTTGAACAGACCCTCGTCTTTATTGTGTGTCATGAGCTGCTAGGGAGCCCTCATCAAAGACCCTTTGTATCGTTTTCGGACGGTGCTTCGACCGCGACCCCAGGTCAGACGGGACTACCCGCTGAGTTTAAGCATATCAATAAGCGGAGGA
>ASV_0000000013
ATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTTTTTGAACGCAAGTTGCGCCCGAAGCCATTCGGCCGAGGGCACGTCTGCCTGGGCGTCACGCATTGCGTCGCCCCAGACTACGCCTCCCCAACGGGGATGCGTTCGACTGGGGCGGAGAATGGTCTCCCGTGTCGTCGGCGTGGTTGGCCTAAAAAGGAGTCCCCTTCGGCGGACGCACGGCTAGTGGTGGTTGTTAAGGCCTTCGTATCGAGCCGTGTGTCGTTAGCCGCAAGGGAAGCACTCTTTAAAGACCCCAATGTGTCGTCTCGTGACGACGCTTCGACCGCGACCCCAGGTCAGGCGGGACTACCCGCTGAGTTTAAGCATATCAATAAGCGGAGGA
>ASV_0000000014
ATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCTAAGCCATTTGGCCGAGGGCACGCCTGCCTGGGCGTCAATCATCTATTCGTCACCCCAACCTCTGCTCCCCATAAAGGAGCTCGGGTCCTGGTTACGGAAGTTGGCCTCCCGTGGTCTCGAAGCGCGGCTGGCCTAAAATTGAGCATCGGGTTGGTGATCTCCGAGGCACGCGGTGGTTGTTCATTCTTACCTCGTGATGTTGCCCCGGGGCATCTTCCACAAGAAGCTCCACGACCCTAGATACATATCGATGCGACCCCAGGTCAGGCGGGGCCACCCGCTGAATTTAAGCATATCAATAAGCGGAGGA
>ASV_0000000015
ATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCAAGTCTTTGAACGCAAGTTGCGCCCGAAGCCATTAGGCCGAGGGCACGTCTGCCTGGGCGTCACACGTCGTTGCCCCCCCCCAACCCCCTCGGGAGTTGGATGGGACGGATGATGGCCTCCCGTGTGCTCAGTCACGCGGTTGGCATAAATACCAAGTCCTCGGCGACCAACGCCACGACAATCGGTGGTTGTCAAACCTCGGTTTCCTGTCGTGCGCGCGTGTTGATCGAGTGCTTTCTTAAACAATGCGTGTCGATCCGTCGATGCTTACAACGCGACCCCAGGTCAGGCGGGGTTACCCGCTGAATTTAAGCATATCAATAAGCGGAGGA
>ASV_0000000016
ATGCGATACTTGGTGTGAATTGCATAATCCCGAAGTGACGGCACGACCGAACAAAGCCCGAGCGGTAGCGGCGGAGACGTCGTGCCCTCGGAAACGCGACCCCAGGTCAGGCGGGGCCACCCGCTGAGTTTAAGCATATCAATAAGCGGAGGA
>ASV_0000000017
ATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCATTAGGCTGAGGGCACGTCTGCCTGGGCGTCACGCATGACGTCGCCCCCCAACCTCGCTCTCACTCGTGGGAGTTGTTGCGGAGGGGCGGATACTGGCCTCCCGTGCCTCATCGTATGGTTGGCCCAAATGTGAGTCCTTGGCGACGGACGTCACGACAAGTGGTGGTTGTAAAAAGCCCTCTTCTCCTGTCGTGCGGTGGCGCGTCGCCAGCAAGAACTCTCGTGACCCTGTTGTGCCGTTGTCAACGCGCACTCCGACTGCGACCCCAGGTCAGGCGGGACTACCCGCTGAGTTTAAGCATATCAATAAGCGGAGGA
>ASV_0000000018
ATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCCAAGCCTTCTGGCCGAGGGCACGTCTGCCTGGGTGTCACAAAAGCTCTCGATGACCCAAAGTCCTCAAAGCGACCCCAGGTCAGGCGGGATCACCCGCTGAGTTTAAGCATATCAATAAGCGGAGGA
>ASV_0000000019
AGGCGATACTTGGTGTGAATGACCGTCACCTTAGTTAGCTCAACGACCCTTACACGCCACAAACTTTGTGCGCTTCGATTGTGACCCCATGTCAGGCGGGATTACCCGCTGAGTTTAAGCATATCAATAAGCGGAGGA
>ASV_0000000020
ATGCGATACTTGGTGTGAATTGAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCTGAAGCCATCAGGTTGAAGGCACGTCTGCCTGGGTGTCACAAATCGTCGTCCCCCCCATCCTCTCGAGGATATGGGACGGAAGCTGGTCTCCCGTGTGTTACCGCACGCGGTTGGCCAAAATCCGAGCTAAGGATGCCAGGAGCGTCTTGACATGCGGTGGTGAATTCAATCTCCTCGTCATATCGTCGGTCGTTCCGGTCCAAAAGCTCTCGATGACCCAAAGTCCTCAACGTGACCCCAGGTCAGGCGGGATCACCCGCTGAGTTTAAGCATATCAATAAGCGGAGGA
>ASV_0000000021
ATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCATTAGGTCGAGGGCACGCCTGCCTGGGCGTCACGCATCGCGTCTCCCCCCAACCACCCTGCGTGGATTGGGAGGAGGATGATGGCCTCCCATGCCTCACCGGGCGTGGATGGCCTAAATAAGGAGCCCCCGGTTACGAAGTGCCGCGGCGATTGGTGGAATACAAGGCCTAGCCTAGGACGAAATCGAAGTCGCGCACATCGTAGCTCTTGAGGACTCGCAGGACCCTAACTTGTTTGCCCCTAGGGGCGGCAAAACCGTTGCGACCCCAGGTCAGGCGGGGCTACCCGCTGAGTTTAAGCATATCAATAAGCGGAGGA
ASVs_taxonomy.txt
ASV_0000000001 k__Viridiplantae; p__Streptophyta; c__Magnoliopsida; o__Brassicales; f__Brassicaceae; g__Brassica; s__Brassica_nigra 0.79
ASV_0000000002 k__Viridiplantae; p__Streptophyta; c__Magnoliopsida; o__Ericales; f__Ericaceae; g__Calluna; s__Calluna_vulgaris 1.0
ASV_0000000003 k__Viridiplantae; p__Streptophyta; c__Magnoliopsida; o__Ericales; f__Ericaceae; g__Erica; s__Erica_cinerea 1.0
ASV_0000000004 k__Viridiplantae; p__Streptophyta; c__Magnoliopsida; o__Rosales; f__Rosaceae; g__Rubus 1.0
ASV_0000000005 k__Viridiplantae; p__Streptophyta; c__Magnoliopsida; o__Asterales; f__Asteraceae; g__Scorzoneroides; s__Scorzoneroides_autumnalis 1.0
ASV_0000000006 k__Viridiplantae; p__Streptophyta; c__Magnoliopsida; o__Fabales; f__Fabaceae; g__Trifolium; s__Trifolium_repens 1.0
ASV_0000000007 k__Viridiplantae; p__Streptophyta; c__Magnoliopsida; o__Fabales; f__Fabaceae; g__Trifolium; s__Trifolium_pratense 1.0
ASV_0000000008 k__Viridiplantae; p__Streptophyta; c__Magnoliopsida; o__Fabales; f__Fabaceae; g__Vicia; s__Vicia_faba 1.0
ASV_0000000009 k__Fungi; p__Ascomycota; c__Saccharomycetes; o__Saccharomycetales; f__Saccharomycetaceae; g__Zygosaccharomyces; s__Zygosaccharomyces_mellis 1.0
ASV_0000000010 k__Viridiplantae; p__Streptophyta; c__Magnoliopsida; o__Lamiales; f__Plantaginaceae; g__Plantago; s__Plantago_lanceolata 1.0
ASV_0000000011 k__Viridiplantae; p__Streptophyta; c__Magnoliopsida; o__Brassicales; f__Brassicaceae; g__Sinapis; s__Sinapis_alba 1.0
ASV_0000000012 k__Viridiplantae; p__Streptophyta; c__Magnoliopsida; o__Asterales; f__Asteraceae; g__Hypochaeris; s__Hypochaeris_radicata 0.98
ASV_0000000013 k__Viridiplantae; p__Streptophyta; c__Magnoliopsida; o__Asterales; f__Asteraceae; g__Cirsium; s__Cirsium_arvense 1.0
ASV_0000000014 k__Viridiplantae; p__Streptophyta; c__Magnoliopsida; o__Myrtales; f__Onagraceae; g__Chamaenerion; s__Chamaenerion_angustifolium 1.0
ASV_0000000015 k__Viridiplantae; p__Streptophyta; c__Magnoliopsida; o__Rosales; f__Rosaceae; g__Rosa 0.81
ASV_0000000016 k__Viridiplantae; p__Streptophyta; c__Magnoliopsida; o__Asparagales; f__Asphodelaceae; g__Phormium; s__Phormium_cookianum 0.5
ASV_0000000017 k__Viridiplantae; p__Streptophyta; c__Magnoliopsida; o__Apiales; f__Araliaceae; g__Hedera; s__Hedera_helix 0.98
ASV_0000000018 k__Viridiplantae; p__Streptophyta; c__Magnoliopsida; o__Ranunculales; f__Menispermaceae; g__Tinospora; s__Tinospora_malabarica 0.5
ASV_0000000019 k__Viridiplantae; p__Streptophyta; c__Magnoliopsida; o__Apiales; f__Apiaceae; g__Conopodium; s__Conopodium_majus 0.74
ASV_0000000020 k__Viridiplantae; p__Streptophyta; c__Magnoliopsida; o__Brassicales; f__Brassicaceae; g__Raphanus; s__Raphanus_sativus 0.7
ASV_0000000021 k__Viridiplantae; p__Streptophyta; c__Magnoliopsida; o__Caryophyllales; f__Chenopodiaceae; g__Atriplex; s__Atriplex_patula 0.77
summary.log
Number of reads: 10441
Number of joined reads: 9379
Number of ASVs (after dada2): 21
Number of ASVs (after ITSx): 18
You need two sets of results from HONEYPI.
First honeypi results:
ASVs_1.fasta
ASVs_counts_1.txt
ASVs_taxonomy_1.txt
Second honeypi results to be added to the first:
ASVs_2.fasta
ASVs_counts_2.txt
ASVs_taxonomy_2.txt
Once you have these files ready, then execute the following commands - watch out for the order of files (they need to be in the precise order)
mamba activate honeypi_env
honeypi_joinTwoResults.py -i1 ASVs_1.fasta,ASVs_counts_1.txt,ASVs_taxonomy_1.txt -i2 ASVs_2.fasta,ASVs_counts_2.txt,ASVs_taxonomy_2.txt
This will produce three files:
ASVs_combined.fasta
ASVs_counts_combined.txt
ASVs_taxonomy_combined.txt