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🧹🦷aKmerBroom: Ancient oral DNA decontamination using Bloom filters on k-mer sets

Read paper here

aKmerBroomis a tool to decontaminate ancient oral samples from a FASTA/FASTQ file. It does so in the following steps:

  1. Build an ancient_kmers.bloom filter from an ancient kmers text file (if such a Bloom filter does not yet exist).
  2. For a set of input reads:
    1. Save those reads which have 2 consecutive kmer matches against ancient_kmers.bloom
    2. Kmerize the saved reads to generate a new set of ancient kmers, called "anchor kmers"
  3. For the same set of input reads, identify matches against anchor kmers and classify each read with >50% matches as an ancient read.

pipeline_svg.pngaKmerBroom pipeline: First, an offline step is performed: a collection of samples representative from diverse sources is used to create a trusted set of oral kmers. The trusted collection indexes kmers that appear exclusively in modern and ancient oral samples, but not other samples from contaminant sources (see panel on the left called Collection of datasets). Then this set of oral kmers is used to decontaminate an input set of reads. The algorithm proceeds by looking up each read kmer inside the Bloom Filter of trusted oral kmers, and marking positions of matches. Reads having at least two consecutive matches to the Bloom Filter get passed to the construction of a set containing all kmers from such reads. Finally, the same input reads are scanned again using the aforementioned set, and reads having a proportion of kmer matches over a certain threshold are reported to be of ancient oral origin.

Usage

# Use the ancient kmers bloom filter provided
python akmerbroom.py --ancient_bloom

or    

# Use an ancient kmers text file 
python akmerbroom.py --ancient_kmers_set

Input

The data/ folder should contain the following input files:

ancient_kmers.bloom : a bloom filter with ancient kmers
unknown_reads.fastq : a file with reads which we want to classify as ancient or not
[optional] ancient_kmers : a text file where each row is a known ancient kmer
[optional] n_consec_matches : Number of consecutive matches in the Bloom Filter that should be used to classify a read as anchor read

Output

The output/ folder should contain the following output files:

annotated_reads.fastq                     # intermediate output
annotated_reads_with_anchor_kmers.fastq   # final output

The final output file has the following 4 fields in each record header:

SeqId, ReadLen, isConsecutiveMatchFound, AnchorProportion

By default, reads with AnchorProportion >= 0.5 (ie. 50%) are chosen as ancient reads.

Dependencies

pip install biopython
pip install cython
pip install pybloomfiltermmap3

Testing

The tests/ folder contains a test dataset consisting of aOral data @SRR13355797 mixed with non aOral data @ERR671934. To run a test, use the following steps:

First, link the test dataset in the input data/ folder:

cd data/
ln -sf ../tests/unknown_reads.fastq .

Next, download the Bloom Filter into the data/ folder from the following link ancient_bloom. Note that it could take a few minutes (file size = 3Gb). This can be done from the command line using the wget utility.

cd data/             # if you are not already in the data/ directory 
wget https://zenodo.org/record/7587160/files/ancient_kmers.bloom -O ancient_kmers.bloom

NOTE: This Bloom Filter was pre-constructed to reduce the running time and facilitate things for users, and it should be used to decontaminate ancient oral samples only. The way it was constructed was detailed on the manuscript for aKmerBroom, but it basically consists of clean k-mers that are of oral origin.

Finally, run aKmerBroom

cd ../              # if you are not already in the main directory
python akmerbroom.py --ancient_bloom

The ancient reads file will be written to output/annotated_reads_with_anchor_kmers.fastq.