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

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.