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Data and methods for PopMLST, a high-resolution method to detect pathogen strain-level diversity in clinical samples.

logo

The publication for this method is in press, and the citation will appear here shortly.

Guide for Use

Installation - Choose a Method:

This software has been tested on the Ubuntu Linux 20.04 operating system. It may be possible to adapt it for other operating systems. We provide two different methods you can use for installation.

Bioconda Method (preferred)

We assume Bioconda is already properly configured and working, per the instructions.

$ conda create -y -n popmlst python=3.8 bioconductor-dada2=1.22.0 vsearch=2.14.0 blast=2.12.0 pandas=1.3.4 biopython=1.79 cutadapt=3.5 pigz=2.6 colorama=0.4.4
$ conda activate popmlst
$ sudo apt update && sudo apt install libtre5
$ wget https://github.com/marade/PopMLST/raw/master/tre-python3.tar.gz && tar xzvf tre-python3.tar.gz && cd tre-python3/python3 && python3 setup.py install && cd ../../ && rm -rf tre-python*

Manual method

The following software dependencies were used for the paper. Other versions may work but have not been tested:

  • Python 3.8.12
  • Python libraries:
    • Biopython 1.79
    • Pandas 1.3.4
    • colorama 0.4.4
    • tre 0.8.0 (see below)
  • Cutadapt 3.5
  • VSEARCH 2.14.0
  • pigz 2.6
  • R 4.1.1
  • DADA2 1.22.0

These are the versions used for the paper, though other versions may work.

The tre Python library hasn't been formally updated for Python 3.x, but community-contributed patches are available. For your convenience we provide a version of the tre library patched for Python 3.x. You can install it like this:

$ wget https://github.com/marade/PopMLST/raw/master/tre-python3.tar.gz
$ tar -xzvf tre-python3.tar.gz
$ cd tre-python/python3
$ python3 setup.py install

Do Sequencing and Generate Fastq Files

We assume you have generated your sequencing data in roughly the manner described in the paper, using Illumina paired-end sequencing. We provide some example files for testing below.

Prepare Fastq Files

This pipeline assumes your paired-end Fastq files are named like so:

sampleX_1.fastq.gz sampleX_2.fastq.gz
sampleY_1.fastq.gz sampleY_2.fastq.gz

Run the Pipeline

Below are instructions for two simple runs using example data for Pseudomonas aeruginosa and Staphylococcus aureus.

$ git clone https://github.com/marade/PopMLST.git
$ cd PopMLST

# run Pseudomonas data
$ python3 ProcessAmpliconData data/Pa PA-cutadapt.tab PA-results
$ Rscript DADA2-PA.R PA-results
$ python3 ParseDADA2Tabs ./ DADA2-PA out.tab D2-PA-combined.tab
$ python3 ParseDADA2Tab -f D2-PA-combined.tab PA-ref D2-PA-table.tab D2-PA-blast.tab
$ python3 FilterDADA2Tab D2-PA-table.filt.tab D2-PA-table.filt2.tab
$ python3 SortColNames D2-PA-table.filt2.tab D2-PA-table.filt.sorted.tab

# run Staph data
$ python3 ProcessAmpliconData data/Sa SA-cutadapt.tab SA-results
$ Rscript DADA2-SA.R SA-results
$ python3 ParseDADA2Tabs ./ DADA2-SA out.tab D2-SA-combined.tab
$ python3 ParseDADA2Tab -f D2-SA-combined.tab SA-ref D2-SA-table.tab D2-SA-blast.tab
$ python3 FilterDADA2Tab D2-SA-table.filt.tab D2-SA-table.filt2.tab
$ python3 SortColNames D2-SA-table.filt2.tab D2-SA-table.filt.sorted.tab

The resulting D2-PA-table.filt.sorted.tab file should look like this:

acs_4 acs_6 aro_5 aro_5* aro_75 gua_11 gua_16 mut_12 mut_3 nuo_1 nuo_4 pps_23 pps_6 trp_1 trp_3
LES100sub 0 11264 0 17 0 19523 0 0 8338 0 19069 21683 0 6311 0
PaCont2sub 19879 1572 94 0 3402 1931 25718 16812 1274 34964 3880 395 4743 567 5840
ASV GGCCCGTTGGCCAACGGCGCCACCACCATTCTGTTCGAGGGCGTACCGAACTACCCCGACGTGACCCGCGTGGCGAAGATCATCGACAAGCACAAGGTTAACATCCTCTACACCGCGCCGACCGCGATCCGCGCGATGATGGCCGAAGGCAAGGCGGCGGTGGCCGGTGCCGACGGTTCCAGCCTGCGTCTGCTCGGTTCGGTGGGCGAGCCGATCAACCCGGAAGCCTGGCAGTGGTACTACGAGACCGTCGGCCAGTCGCGCTGCCCGATCGTCGACACCTGGTGGCAGACCGAGACCGGCGCCTGCCTGATGACCCCGTTGCCGGGCGCCCATGCGATGAAGCCGGGCTCCGCGGCCAAGCCGTTCTTCGGCGTGGTCCCGGCGCTG GGCCCGTTGGCCAACGGCGCCACCACCATTCTGTTCGAGGGCGTGCCGAACTACCCCGACGTGACCCGCGTGGCGAAAATCATCGACAAGCACAAGGTCAACATCCTCTACACCGCGCCGACCGCGATCCGCGCGATGATGGCCGAAGGCAAGGCGGCGGTGGCCGGTGCCGACGGTTCCAGCCTGCGTCTGCTCGGTTCGGTGGGCGAGCCGATCAACCCGGAAGCCTGGCAGTGGTACTACGAGACCGTCGGCCAGTCGCGCTGCCCGATCGTCGACACCTGGTGGCAGACCGAGACCGGCGCCTGCCTGATGACCCCGTTGCCGGGCGCCCATGCGATGAAGCCGGGCTCCGCGGCCAAGCCGTTCTTCGGCGTGGTCCCGGCGCTG ATGTCACCGTGCCGTTCAAGGAAGAGGCCTATCGTCTGGTGGACGAGTTGAGCGAGCGGGCCACCCGGGCCGGGGCGGTGAACACCCTGATCCGCCTCGCCGACGGTCGCCTGCGCGGCGACAACACCGACGGCGCCGGCCTGCTGCGGGACCTGACGGCGAACGCCGGGGTCGAGCTGCGCGGCAAGCGGGTTCTCCTGCTCGGCGCCGGCGGTGCGGTGCGTGGGGTGCTCGAACCCTTCCTCGGCGAGTGCCCGGCGGAGTTGCTGATCGCCAACCGCACGGCGCGGAAGGCCGTGGACCTGGCCGAGCGGTTCGCCGACCTCGGCGCGGTGCACGGCTGCGGTTTCGCCGAGGTCGAAGGGCCTTTCGACCTGATCGTCAACGGCACCTCGGCCAGTCTTGCCGGCGACGTGCCGCCGCTGGCGCAGAGCGTGATCGAGCCCGGCCGTACCGTCTGCTACGACATGATGTATGCCAAGGAACCGACTGCCTTCA ATGTCACCGTGCCGTTCAAGGAAGAGGCCTATCGTCTGGTGGACGAGTTGAGCGAGCGGGCCACCCGGGCCGGGGCGGTGAACACCCTGATCCGCCTCGCCGACGGTCGCCTGCGCGGCGACAACACCGACGGCGCCGGCCTGCTGCGGGACCTGACGGCGAACGCCGGGGTCGAGCTGCGCGGCAAGCGGGGTCTCCTGCTCGGCGCCGGCGGTGCGGTGCGTGGGGTGCTCGAACCCTTCCTCGGCGAGTGCCCGGCGGAGTTGCTGATCGCCAACCGCACGGCGCGGAAGGCCGTGGACCTGGCCGAGCGGTTCGCCGACCGCGGCGCGGTGCACGGCTGCGGTTTCGCCGAGGTCGAAGGGCCTTTCGACCTGATCGTCAACGGCACCTCGGCCAGTCTTGCCGGCGACGTGCCGCCGCTGGCGCAGAGCGTGATCGAGCCCGGCCGTACCGTCTGCTACGACATGATGTATGCCAAGGAACCGACTGCCTTCA ATGTCACCGTGCCGTTCAAGGAAGAGGCCTATCGTCTGGTGGACGAATTGAGCGAGCGGGCCACCCGGGCCGGGGCGGTGAACACCCTGATCCGCCTGGCCGACGGTCGCCTGCGCGGCGACAACACCGACGGCGCGGGCTTGCTGCGGGACCTGACGGCGAACGCCGGGGTCGAGCTGCGCGGCAAGCGGGTTCTCCTGCTCGGCGCCGGCGGTGCGGTGCGCGGGGTGCTCGAACCCTTCCTCGGCGAGTGCCCGGCGGAGTTGCTGATCGCCAACCGCACGGCGCGGAAGGCCGTGGACCTGGCCGAGCGATTCGCCGATCTCGGCGCGGTGCGCGGCTGCGGTTTCGCCGAGGTCGAAGGGCCTTTCGACCTGGTCGTCAACGGCACCTCGGCCAGTCTTGCCGGCGACGTGCCGCCGCTGGCGCAGAGCGTGATCGAGCCCGGCCGTACCGTCTGCTACGACATGATGTATGCCAAGGAACCGACTGCCTTCA CTGCTAGGCCTCTCCGGCGGCGTGGACTCCTCGGTGGTCGCCGCGCTGCTGCACAAGGCCATCGGCGACCAACTGACCTGCGTGTTCGTCGACAACGGCCTGCTGCGCCTGCACGAAGGCGACCAGGTGATGGCCATGTTCGCCGAGAACATGGGCGTGAAGGTGATCCGCGCCAACGCCGAGGACAAGTTCCTCGGCCGCCTGGCCGGCGTCGCCGACCCGGAAGAGAAGCGCAAGATCATCGGCCGCACCTTCATCGAAGTTTTCGACGAAGAAGCCACCAAGCTGCAGGACGTGAAGTTCCTCGCCCAGGGCACCATCTACCCCGACGTGATCGAGTCGGCCGGCGCCAAGACCGGCAAGGCCCACGTGA CTGCTCGGCCTCTCCGGCGGCGTGGACTCCTCGGTGGTCGCCGCGCTGCTGCACAAGGCCATCGGCGACCAACTGACCTGCGTGTTCGTCGACAACGGCCTGCTGCGCCTGCACGAAGGCGACCAGGTGATGGCCATGTTCGCCGAGAACATGGGCGTGAAGGTGATCCGCGCCAACGCCGAGGACAAGTTCCTCGGCCGCCTGGCCGGCGTCGCCGATCCGGAAGAGAAGCGCAAGATCATCGGCCGCACCTTCATCGAAGTCTTCGACGAAGAAGCCACCAAGCTGCAGGACGTGAAGTTCCTCGCCCAGGGCACCATCTACCCCGACGTGATCGAGTCGGCCGGCGCCAAAACCGGCAAGGCCCACGTGA CTGCAGGAAGTCATCAAGCGCCTGGCGCTGGCCCGTTTCGACGTGGCTTTCCACCTGCGCCACAACGGCAAGACCATCTTCGCCCTGCACGAGGCGCGAGACGAGCTGGCCCGCGCGCGCCGGGTCGGCGCGGTGTGCGGCCAGGCATTCCTCGAGCAGGCGCTGCCGATCGAGGTCGAGCGCAACGGCCTGCACCTGTGGGGCTGGGTCGGCTTGCCGACCTTCTCCCGCAGCCAGCCGGACCTGCAGTACTTCTATGTGAACGGGCGCATGGTGCGCGACAAGCTGGTCGCCCACGCGGTGCGCCAGGCTTATCGCGACGTGCTGTACAACGGCCGGCACCCGACCTTCGTGCTGTTCTTCGAAGTCGATCCGGCGGTGGTGGACGTCAACGTGCACCCGACCAAGCACGAAGTTCGCTTCCGTGACAGCCGGATGGTCC CTGCAGGAGGTCATCAAGCGCCTGGCGCTGGCCCGCTTCGACGTGGCTTTCCACCTGCGCCACAACGGCAAGACCATCTTCGCCCTGCACGAGGCGCGAGACGAGCTGGCCCGCGCGCGCCGGGTCGGCGCGGTGTGCGGCCAGGCATTCCTCGAGCAGGCGCTGCCGATCGAGGTCGAGCGCAACGGCCTGCACCTGTGGGGCTGGGTCGGCTTGCCGACCTTCTCCCGCAGCCAGCCGGACCTGCAGTACTTCTATGTGAACGGGCGCATGGTGCGCGACAAGCTGGTCGCCCACGCGGTGCGCCAGGCTTATCGCGACGTGCTGTACAACGGCCGGCATCCGACCTTCGTGCTGTTCTTCGAAGTCGATCCGGCGGTGGTGGACGTCAACGTGCACCCGACCAAGCACGAAGTTCGCTTCCGTGACAGCCGGATGGTCC ATGTTCCTCAACCTCGGCCCGAACCACCCGTCCGCCCACGGCGCGTTCCGCATCATCCTGCAACTGGACGGCGAGGAGATCATCGACTGCGTCCCGGAGATCGGCTACCACCACCGCGGCGCCGAGAAGATGGCCGAGCGCCAGTCCTGGCACAGTTTCATCCCCTACACCGACCGCATCGACTACCTCGGCGGGGTGATGAACAACCTGCCCTACGTACTCTCGGTGGAGAAGCTCGCCGGGATCAAGGTGCCGCAGCGGGTCGACGTGATCCGGATCATGATGGCGGAGTTCTTCCGTATCCTGAACCACCTGCTGTACCTGGGCACCTATATCCAGGACGTCGGCGCCATGACCCCGGTGTTC ATGTTCCTCAACCTCGGCCCGAACCACCCGTCCGCCCACGGCGCGTTCCGCATCATCCTGCAACTGGACGGCGAGGAGATCATCGACTGCGTCCCGGAGATCGGCTACCACCACCGCGGCGCCGAGAAGATGGCCGAGCGCCAGTCCTGGCACAGTTTCATCCCCTACACCGACCGCATCGACTACCTCGGCGGGGTGATGAACAACCTGCCCTACGTACTCTCGGTGGAGAAGCTCGCCGGGATCAAGGTGCCCCAGCGGGTCGACGTGATCCGGATCATGATGGCGGAGTTCTTCCGTATCCTGAACCACCTGCTGTACCTGGGCACCTATATCCAGGACGTCGGCGCCATGACCCCGGTGTTC CATCGTCCAGGCACGCCCGGAAACCGTGAAGAGCCGCGCCAGCGCCACGGTCATGGAGCGCTACCTGCTGAAAGAGAAGGGGACCGTCCTGGTGGAAGGGCGTGCCATCGGCCAGCGCATCGGTGCCGGTCCGGTCAAGGTGATCAACGACGTGTCGGAAATGGACAAGGTCCAACCGGGTGACGTCCTGGTCTCCGACATGACCGACCCGGACTGGGAGCCGGTGATGAAGCGCGCCAGCGCCATCGTCACCAACCGCGGCGGGCGTACCTGCCACGCGGCGATCATCGCTCGCGAACTGGGCATCCCGGCGGTGTTCGGTTGCGGCAACGCCACCCAGATCCTGCAGGATGGCCAGGGGGTGACCGTT CATCGTCCAGGCACGCCCGGAAACCGTGAAGAGCCGCGCCAGCGCCACGGTCATGGAGCGCTACCTGCTGAAAGAGAAGGGGACCGTCCTGGTGGAAGGACGTGCCATCGGCCAGCGCATCGGTGCCGGTCCGGTCAAGGTGATCAACGACGTGTCGGAAATGGACAAGGTCCAACCGGGTGACGTCCTGGTCTCCGACATGACCGACCCGGACTGGGAGCCGGTGATGAAGCGCGCCAGCGCCATCGTCACCAACCGCGGCGGGCGTACCTGCCACGCGGCGATCATCGCTCGCGAACTGGGCATCCCGGCGGTGGTCGGTTGCGGCAACGCCACCCAGATCCTGCAGGATGGGCAGGGGGTGACCGTT TGTCGTGGGCAGCTCGCCGGAGGTGCTGGTACGGGTCGAGGATGGCCTGGTGACGGTGCGCCCGATCGCCGGTACCCGTCCGCGCGGGATCAACGAAGAGGCCGACCTGGCGCTGGAGCAGGATCTGCTGTCGGACGCCAAGGAGATCGCCGAGCACCTGATGCTGATCGACCTGGGGCGCAACGACGTGGGGCGGGTGTCCGATATCGGCGCGGTGAAGGTCACCGAAAAAATGGTGATCGAACGTTACTCCAACGTCATGCACATCGTGTCCAACGTCACCGGGCAATTGCGCGAGGGGCTCAGCGCGATGGACGCGCTGCGGGCGATTCTGCCGGCGGGCACTCTATCCGGCGCGCCGAAGATCCGCGCCATGGAGATCATCGACGAGCTGGAGCCGGTCAAGCGTGGAGTCTACGGCGGCGCGGTCGGCTACCTGGCAT TGTCGTGGGCAGCTCGCCGGAGGTGCTGGTACGGGTCGAGGATGGCCTGGTGACGGTGCGCCCGATCGCCGGTACCCGTCCGCGCGGGATCAACGAAGAGGCCGACCTGGCGCTGGAGCAGGATCTGCTGTCGGACGCCAAGGAGATCGCCGAGCACCTGATGCTGATCGACCTGGGGCGCAACGACGTGGGGCGGGTGTCCGACATCGGCGCGGTGAAGGTCACCGAAAAAATGGTGATCGAACGTTACTCCAACGTCATGCACATCGTGTCCAACGTCACCGGGCAATTGCGCGAGGGGCTCAGCGCGATGGACGCGCTGCGGGCGATCCTGCCGGCGGGTACGCTGTCCGGCGCGCCGAAGATCCGCGCCATGGAGATCATCGACGAGCTGGAGCCGGTCAAGCGTGGAGTCTACGGCGGCGCGGTCGGCTACCTGGCAT

The resulting D2-SA-table.filt.sorted.tab file should look like this:

arcC_10 arcC_13 arcC_3 aroE_13 aroE_14 aroE_3 glpF_1 glpF_8 gmk_1 gmk_6 pta_10 pta_12 pta_4 tpi_11 tpi_3 tpi_4 yqiL_13 yqiL_2 yqiL_3
2-8 0 10933 0 13163 0 0 11680 0 17466 0 0 9385 0 19384 0 0 13791 0 0
No-36-33-10-1A 3790 0 921 0 6148 341 357 3278 1594 9787 5867 0 962 0 13174 2309 0 5441 1033
ASV TTATTAATCCAACAAGCTAAATCGAACAGTGACACAACGCCGGCAATGCCATTGGATACTTGTGGTGCAATGTCACAGGGTATGATAGGCTATTGGTTGGAAACTGAAATCAATCGCATTTTAACTGAAATGAATAGTGATAGAACTGTAGGCACAATCGTAACACGTGTGGAAGTAGATAAAGATGATCCACGATTCAATAACCCAACCAAACCAATTGGTCCTTTTTATACGAAAGAAGAAGTTGAAGAATTACAAAAAGAACAGCCAGACTCAGTCTTTAAAGAAGATGCAGGACGTGGTTATAGAAAAGTAGTTGCGTCACCACTACCTCAATCTATACTAGAACACCAGTTAATTCGAACTTTAGCAGACGGTAAAAATATTGTCATTGCATGCGGTGGTGGCGGTATTCCAGTTATAAAAAAAGAAAATACCTATGAAGGTGTTGAAGCG TTATTAATCCAACAAGCTAAATCGAACAGTGACACAACGCCGGCAATGCCATTGGATACTTGTGGTGCAATGTCACAGGGTATGATAGGCTATTGGTTGGAAACTGAAATCAATCGCATTTTAACTGAAATGAATAGTGATAGAACTGTAGGCACAATCGTTACACGTGTGGAAGTAGATAAAGATGATCCACGATTCAATACCCCAACCAAACCAATTGGTCCTTTTTATACGAAAGAAGAAGTTGAAGAATTACAAAAAGAACAGCCAGACTCAGTCTTTAAAGAAGATGCAGGACGTGGTTATAGAAAAGTAGTTGCGTCACCACTACCTCAATCTATACTAGAACACCAGTTAATTCGAACTTTAGCAGACGGTAAAAATATTGTCATTGCATGCGGTGGTGGCGGTATTCCAGTTATAAAAAAAGAAAATACCTATGAAGGTGTTGAAGCG TTATTAATCCAACAAGCTAAATCGAACAGTGACACAACGCCGGCAATGCCATTGGATACTTGTGGTGCAATGTCACAGGGTATGATAGGCTATTGGTTGGAAACTGAAATCAATCGCATTTTAACTGAAATGAATAGTGATAGAACTGTAGGCACAATCGTTACACGTGTGGAAGTAGATAAAGATGATCCACGATTTGATAACCCAACTAAACCAATTGGTCCTTTTTATACGAAAGAAGAAGTTGAAGAATTACAAAAAGAACAGCCAGACTCAGTCTTTAAAGAAGATGCAGGACGTGGTTATAGAAAAGTAGTTGCGTCACCACTACCTCAATCTATACTAGAACACCAGTTAATTCGAACTTTAGCAGACGGTAAAAATATTGTCATTGCATGCGGTGGTGGCGGTATTCCAGTTATAAAAAAAGAAAATACCTATGAAGGTGTTGAAGCG AATTTTAATTCTTTAGGATTAGCTGATACTTATGAAGCTTTAAATATTCCAATTGAAGATTTTCATTTAATTAAAGAAATTATTTCAAAAAAAGAATTAGATGGCTTTAATATCACAATTCCTCATAAAGAACGTATCATATCGTATTTAGATCATGTTGATGAACAAGCGATTAATGCAGGTGCAGTTAACACTGTTTTGATAAAAGATGGCAAGTGGATAGGGTATAATACAGATGGTATTGGTTATGTTAAAGGATTGCACAGCGTTTATCCAGATTTAGAAAATGCATACATTTTAATTTTGGGCGCAGGTGGTGCAAGTAAAGGCATTGCTTATGAATTAGCAAAATTTGTAAAGCCCAAATTAACTGTTGCGAATAGAACGATGGCTCGTTTTGAATCTTGGAATTTAAATATAAACCAAATTTCATTAGCAGATGCTGAAAAGTATTTA AATTTTAATTCTTTGGGATTAGATGATACTTATGAAGCTTTAAATATTCCAATTGAAGATTTTCATTTAATTAAAGAAATTATTTCAAAAAAAGAATTAGATGGCTTTAATATCACAATTCCTCATAAAGAGCGTATCATACCGTATTTAGATCATGTTGATGAACAAGCGATTAATGCAGGTGCAGTTAATACTGTTTTGATAAAAGATGGCAAGTGGATAGGGTATAATACAGATGGTATTGGTTATGTAAAAGGATTGCACAGCGTTTATCCAGATTTAGAAAATGCATACATTTTAATTTTGGGAGCAGGTGGTGCAAGTAAAGGTATTGCTTATGAATTAGCAAAATTTGTAAAGCCCAAATTAACTGTTGCGAATAGAACGATGGCTCGTTTTGAATCTTGGAATTTAAATATAAACCAAATTTCATTGGCAGATGCTGAAAAGTATTTA AATTTTAATTCTTTAGGATTAGATGATACTTATGAAGCTTTAAATATTCCAATTGAAGATTTTCATTTAATTAAAGAAATTATTTCGAAAAAAGAATTAGAAGGCTTTAATATCACAATTCCTCATAAAGAACGTATCATACCGTATTTAGATTATGTTGATGAACAAGCGATTAATGCAGGTGCAGTTAACACTGTTTTGATAAAAGATGGCAAGTGGATAGGGTATAATACAGATGGTATTGGTTATGTTAAAGGATTGCACAGCGTTTATCCAGATTTAGAAAATGCATACATTTTAATTTTGGGCGCAGGTGGTGCAAGTAAAGGTATTGCTTATGAATTAGCAAAATTTGTAAAGCCCAAATTAACTGTTGCGAATAGAACGATGGCTCGTTTTGAATCTTGGAATTTAAATATAAACCAAATTTCATTAGCAGATGCTGAAAAGTATTTA GGTGCTGATTGGATTGTCATCACAGCTGGATGGGGATTAGCGGTTACAATGGGTGTGTTTGCTGTCGGTCAATTCTCAGGTGCACATTTAAACCCAGCGGTGTCTTTAGCTCTTGCATTAGACGGAAGTTTTGATTGGTCATTAGTTCCTGGTTATATTGTTGCTCAAATGTTAGGTGCAATTGTCGGAGCAACAATTGTATGGTTAATGTACTTGCCACATTGGAAAGCGACAGAAGAAGCTGGCGCGAAATTAGGTGTTTTCTCTACAGCACCGGCTATTAAGAATTACTTTGCCAACTTTTTAAGTGAGATTATCGGAACAATGGCATTAACTTTAGGTATTTTATTTATCGGTGTAAACAAAATTGCCGATGGTTTAAATCCTTTAATTGTCGGAGCATTAATTGTTGCAATCGGATTAAGTTTAGGCGGTGCTACTGGTTATGCAATCAACCCAGCACGT GGTGCTGATTGGATTGTCATCACAGCTGGATGGGGATTAGCGGTTACAATGGGTGTATATGCTGTCGGTCAATTCTCAGGTGCACATTTAAACCCAGCGGTGTCTTTAGCTCTTGCATTAGACGGAAGTTTTGATTGGTCATTAGTTCCTGGTTATATTGTTGCTCAAATGTTAGGTGCAATTGTCGGAGCAACGATTGTATGGTTAATGTACTTGCCACATTGGAAAGCGACAGAAGAAGCTGGCGCGAAATTAGGTGTTTTCTCTACAGCACCGGCTATTAAGAATTACTTTGCCAACTTTTTAAGTGAGATTATCGGAACAATGGCATTAACTTTAGGTATTTTATTTATCGGTGTAAACAAAATTGCCGATGGTTTAAATCCTTTAATTGTCGGAGCATTAATTGTTGCAATTGGATTAAGTTTAGGCGGTGCTACTGGTTATGCAATCAACCCAGCACGT CGAATATTTGAAGATCCAAGTACATCATATAAGTATTCTATTTCAATGACAACACGTCAAATGCGTGAAGGTGAAGTTGATGGCGTAGATTACTTTTTTAAAACTAGGGATGCGTTTGAAGCTTTAATCAAAGATGACCAATTTATAGAATATGCTGAATATGTAGGCAACTATTATGGTACACCAGTTCAATATGTTAAAGATACAATGGACGAAGGTCATGATGTATTTTTAGAAATTGAAGTAGAAGGTGCAAAGCAAGTTAGAAAGAAATTTCCAGATGCGCTATTTATTTTCTTAGCACCTCCAAGTTTAGAACACTTGAGAGAGCGATTAGTAGGTAGAGGAACAGAATCTGATGAGAAAATACAAAGTCGTATTAACGAAGCGCGTAAAGAAGTTGAAATGATGAATTTA CGAATATTTGAAGATCCAAGTACATCATATAAGTATTCTATTTCAATGACAACACGTCAAATGCGTGAAGGTGAAGTTGATGGCGTAGATTACTTTTTTAAAACTAGGGATGCGTTTGAAGCTTTAATTAAAGATGACCAATTTATAGAATATGCTGAATATGTAGGCAACTATTATGGTACACCAGTTCAATATGTTAAAGATACAATGGACGAAGGTCATGATGTATTTTTAGAAATTGAAGTAGAAGGTGCAAAGCAAGTTAGAAAGAAATTTCCAGATGCGTTATTTATTTTCTTAGCACCTCCAAGTTTAGATCACTTGAGAGAGCGATTAGTAGGTAGAGGAACAGAATCTGATGAGAAAATACAAAGTCGTATTAACGAAGCACGTAAAGAAGTTGAAATGATGAATTTA GCAACACAATTACAAGCAACAGATTATGTTACACCAATCGTGTTAGGTGATGAGACTAAGGTTCAATCTTTAGCGCAAAAACTTAATCTTGATATTTCTAATATTGAATTAATTAATCCTGCGACAAGTGAATTGAAAGCTGAATTAGTTCAATCATTTGTTGAACGACGTAAAGGTAAAGCGACTGAAGAACAAGCACAAGAATTATTAAACAATGTGAACTACTTCGGTACAATGCTTGTTTATGCTGGTAAAGCAGATGGTTTAGTTAGTGGTGCAGCACATTCAACAGGCGACACTGTGCGTCCAGCATTACAAATCATCAAAACGAAACCAGGTGTATCAAGAACATCAGGTATCTTCTTTATGATTAAAGGTGATGAACAATACATCTTTGGTGATTGTGCAATCAATCCAGAACTTGATTCACAAGGACTTGCAGAAATTGCAGTAGAAAGTGCAAAATCAGCATTA GCAACACAATTACAAGCAACAGATTATGTTACACCAATCGTGTTAGGTGATGAGACTAAGGTTCAATCTTTAGCGCAAAAACTTGATCTTGATATTTCTAATATTGAATTAATTAATCCTGCGACAAGTGAATTGAAAGCTGAATTAGTTCAATCATTTGTTGAACGACGTAAAGGTAAAGCGACTGAAGAACAAGCACAAGAATTATTAAACAATGTGAACTACTTCGGTACAATGCTTGTTTATGCTGGTAAAGCAGATGGTTTAGTTAGTGGTGCAGCACATTCAACAGGAGACACTGTGCGTCCAGCTTTACAAATCATCAAAACGAAACCAGGTGTATCAAGAACATCAGGTATCTTCTTTATGATTAAAGGTGATGAACAATACATCTTTGGTGATTGTGCAATCAATCCAGAACTTGATTCACAAGGACTTGCAGAAATTGCAGTAGAAAGTGCAAAATCAGCATTA GCAACACAATTACAAGCAACAGATTATGTTACACCAATCGTGTTAGGTGATGAGACTAAGGTTCAATCTTTAGCGCAAAAACTTGATCTTGATATTTCTAATATTGAATTAATTAATCCTGCGACAAGTGAATTGAAAGCTGAATTAGTTCAATCATTTGTTGAACGACGTAAAGGTAAAGCGACTGAAGAACAAGCACAAGAATTATTAAACAATGTGAACTACTTCGGTACAATGCTTGTTTATGCTGGTAAAGCAGATGGTTTAGTTAGTGGTGCAGCACATTCAACAGGCGACACTGTGCGTCCAGCTTTACAAATCATCAAAACGAAACCAGGTGTATCAAGAACATCAGGTATCTTCTTTATGATTAAAGGTGATGAACAATACATCTTTGGTGATTGTGCAATCAATCCAGAACTTGATTCACAAGGACTTGCAGAAATTGCAGTAGAAAGTGCAAAATCAGCATTA CACGAAACAGATGAAGAAATTAACAAAAAAGCGCACGCTATTTTCAAATATGGAATGACTCCAATTATTTGTGTTGGTGAAACAGACGAAGAGCGTGAAAGTGGTAAAGCTAACGATGTTGTAGGTGAGCAAGTTAAGAAAGCTGTTGCAGGTTTATCTGAAGATCAACTTAAATCAGTTGTAATTGCTTATGAGCCAATCTGGGCAATCGGAACTGGTAAATCATCAACATCTGAAGATGCAAATGAAATGTGTGCATTTGTACGTCAAACTATTGCTGACTTATCAAGCAAAGAAGTATCAGAAGCAACTCGTATTCAATATGGTGGTAGTGTTAAACCTAACAACATTAAAGAATACATGGCACAAACTGATATTGATGGGGCATTAGTAGGTGGCGCA CACGAAACAGATGAAGAAATTAACAAAAAAGCGCACGCTATTTTCAAACATGGAATGACTCCAATTATTTGTGTTGGTGAAACAGACGAAGAGCGTGAAAGTGGTAAAGCTAACGATGTTGTAGGTGAGCAAGTTAAGAAAGCTGTTGCAGGTTTATCTGAAGATCAACTTAAATCAGTTGTAATTGCTTATGAACCAATCTGGGCAATCGGAACTGGTAAATCATCAACATCTGAAGATGCGAATGAAATGTGTGCATTTGTACGTCAAACTATTGCTGACTTATCAAGCAAAGAAGTATCAGAAGCAACTCGTATTCAATATGGTGGTAGTGTTAAACCTAACAACATTAAAGAATACATGGCACAAACTGATATTGATGGGGCATTAGTAGGTGGCGCA CACGAAACAGATGAAGAAATTAACAAAAAAGCGCACGCTATTTTCAAACATGGAATGACTCCAATTATATGTGTTGGTGAAACAGACGAAGAGCGTGAAAGTGGTAAAGCTAACGATGTTGTAGGTGAGCAAGTTAAGAAAGCTGTTGCAGGTTTATCTGAAGATCAACTTAAATCAGTTGTAATTGCTTATGAACCAATCTGGGCAATCGGAACTGGTAAATCATCAACATCTGAAGATGCAAATGAAATGTGTGCATTTGTACGTCAAACTATTGCTGACTTATCAAGCAAAGAAGTATCAGAAGCAACTCGTATTCAATATGGTGGTAGTGTTAAACCTAACAACATTAAAGAATACATGGCACAAACTGATATTGATGGGGCATTAGTAGGTGGCGCA GCGTTTAAAGACGTGCCAGCCTATGATTTAGGTGCGACTTTAATAGAACATATTATTAAAGAGACGGGTTTGAATCCAAGTGAGATTGATGAAGTTATCATCGGTAACGTACTACAAGCAGGACAAGGACAAAATCCAGCACGAATTGCTGCTATGAAAGGTGGCTTGCCAGAAACAGTACCTGCATTTACAGTGAATAAAGTATGTGGTTCTGGGTTAAAGTCGATTCAATTAGCATATCAATCTATTGTGACTGGTGAAAATGACATCGTGCTAGCTGGCGGTATGGAGAATATGTCTCAGTCACCAATGCTTGTCAACAACAGTCGCTTTGGTTTTAAAATGGGACATCAATCAATGGTTGATAGCATGGTATATGATGGTTTAACAGATGTATTTAATCAATATCATATGGGTATTACTGCTGAAAATTTAGTAGAGCAATATGGTATTTCAAGAGAAGAACAAGATACATTTGCTGTAAACTCACAACAAAAAGCAGTACGTGCACAGCAA GCGTTTAAAGACGTGCCAGCCTATGATTTAGGTGCGACTTTAATAGAACATATTATTAAAGAGACGGGTTTGAATCCAAGTGAGATTAATGAAGTCATCATCGGTAACGTACTACAAGCAGGACAAGGACAAAATCCAGCACGAATTGCTGCTATGAAAGGTGGCTTGCCAGAAACAGTACCTGCATTTACAGTGAATAAAGTATGTGGTTCTGGGTTAAAGTCGATTCAATTAGCATATCAATCTATTGTGACTGGTGAAAATGACATCGTGCTAGCTGGCGGTATGGAGAATATGTCTCAATCACCAATGCTTGTCAACAACAGTCGCTTTGGTTTTAAAATGGGACATCAATCAATGGTTGATAGCATGGTATATGATGGTTTAACAGATGTATTTAATCAATATCATATGGGTATTACTGCTGAAAATTTAGTAGAGCAATATGGTATTTCAAGAGAAGAACAAGATACATTTGCTGTAAACTCACAACAAAAAGCAGTACGTGCACAGCAA GCGTTTAAAGACGTGCCAGCCTATGATTTAGGTGCGACTTTAATAGAACATATTATTAAAGAGACGGGTTTGAATCCAAGTGAGATTGATGAAGTTATCATCGGTAACGTACTACAAGCAGGACAAGGACAAAATCCAGCACGAATTGCTGCTATGAAAGGTGGCTTGCCAGAAACAGTACCTGCATTTACGGTGAATAAAGTATGTGGTTCTGGGTTAAAGTCGATTCAATTAGCATATCAATCTATTGTGACTGGTGAAAATGACATCGTGCTAGCTGGCGGTATGGAGAATATGTCTCAATCACCAATGCTTGTCAACAACAGTCGCTTTGGTTTTAAAATGGGACATCAATCAATGGTTGATAGCATGGTATATGATGGTTTAACAGATGTATTTAATCAATATCATATGGGTATTACTGCTGAAAATTTAGTAGAGCAATATGGTATTTCAAGAGAAGAACAAGATACATTTGCTGTAAACTCACAACAAAAAGCAGTACGTGCACAGCAA

Please consult the paper for more details on this method.