Tutorial

Use Chorus2 to design oligo probes for plant genome

In this tutorial, we will build oligo probe set for Arabidopsis genome.

Install Chorus2

See install tutorial here

Run Chorus2

Run Chorus2 with Docker

Download Reference Genome file

$ wget https://www.arabidopsis.org/download_files/Genes/TAIR10_genome_release/TAIR10_chromosome_files/TAIR10_chr_all.fas

$ docker run -v $PWD:/home/chorus -e CHORUS_USER=$USER -e CHORUS_UID=$UID \
  forrestzhang/docker-chorus -i TAIR10_chr_all.fas -g TAIR10_chr_all.fas -t 12

Please wait unit all precess done. There are some logs:

forrest /home/chorus
use local user:  forrest
Adding group 'forrest' (GID 1000) ...
Done.
Adding user 'forrest' ...
Adding new user 'forrest' (1000) with group 'forrest' ...
Creating home directory '/home/forrest' ...
Copying files from '/etc/skel' ...
/home/chorus exists
2.2.3
########################################
bwa version: /opt/software/bwa/bwa 0.7.12-r1044
jellyfish version: /opt/software/jellyfish/bin/jellyfish 2.2.3
genome file: TAIR10_chr_all.fas
input file: TAIR10_chr_all.fas
5' labeled R primer:
result output folder: /home/chorus/probes
threads number: 12
homology: 75
dtm: 10
########################################
...
...
14300000 / 14326857
14310000 / 14326857
14320000 / 14326857
Job finshed!!

When process done:

$ ls -lt probes/
total 1741428
-rw-r--r-- 1 root root  280927981 Aug 24 17:44 TAIR10_chr_all.fas_all.bed
-rw-r--r-- 1 root root   62050561 Aug 24 17:44 TAIR10_chr_all.fas.bed
-rw-r--r-- 1 root root         94 Aug 24 17:30 TAIR10_chr_all.fas.len
-rw-r--r-- 1 root root 1031512169 Aug 24 17:22 TAIR10_chr_all.fas_tmp_probe.fa
-rw-r--r-- 1 root root   59833928 Aug 24 17:19 TAIR10_chr_all.fas.sa
-rw-r--r-- 1 root root       7535 Aug 24 17:18 TAIR10_chr_all.fas.amb
-rw-r--r-- 1 root root        682 Aug 24 17:18 TAIR10_chr_all.fas.ann
-rw-r--r-- 1 root root   29916939 Aug 24 17:18 TAIR10_chr_all.fas.pac
-rw-r--r-- 1 root root  119667836 Aug 24 17:18 TAIR10_chr_all.fas.bwt
-rw-r--r-- 1 root root  121183059 Aug 24 17:17 TAIR10_chr_all.fas
-rw-r--r-- 1 root root   78102510 Aug 24 17:17 TAIR10_chr_all.fas_17mer.jf

TAIR10_chr_all.fas.bed is the probe file contained non-overlapped probes.

TAIR10_chr_all.fas_all.bed is the probe file contained all probes. This file can be used for ChorusNGSfilter.

TAIR10_chr_all.fas.len is the length info of the given genome chromosomes. This file can be imported into ChorusPBGUI for probe selection.

TAIR10_chr_all.fas_17mer.jf is the binary file created by jellyfish count using 17-mer.

TAIR10_chr_all.fas_tmp_probe.fa contains all candidate probe sequences filtered by jellyfish.

.bwt, .pac, .ann, .amb, .sa files are bwa index files.

$ more probes/TAIR10_chr_all.fas.bed
1           52      96      TCCCTAAATCTTTAAATCCTACATCCATGAATCCCTAAATACCTA
1           211     255     TTTGAGGTCAATACAAATCCTATTTCTTGTGGTTTTCTTTCCTTC
1           346     390     CCTTAGGGTTGGTTTATCTCAAGAATCTTATTAATTGTTTGGACT
1           426     470     TTTGTGGAAATGTTTGTTCTATCAATTTATCTTTTGTGGGAAAAT
1           496     540     TCTTCGTTGTTGTTACGCTTGTCATCTCATCTCTCAATGATATGG
1           551     595     TAGCATTTATTCTGAAGTTCTTCTGCTTGATGATTTTATCCTTAG

There are four columns in each row, first column is chromosome name, second is oligo start site, third is oligo end site, the last one is oligo probe sequence. You can use excel or text editor to open this file.

Run Chorus2 in terminal

Make a project folder

$ cd ~
$ mkdir sampleproject
$ cd sampleproject

Download reference genome

$ wget https://www.arabidopsis.org/download_files/Genes/TAIR10_genome_release/TAIR10_chromosome_files/TAIR10_chr_all.fas

Test chorus2 software

$ Chorus2 -h
usage: Chorus2 [-h] [--version] [-j JELLYFISH] [-b BWA] -g GENOME -i INPUT
            [-s SAVED] [-p PRIMER] [-t THREADS] [-l LENGTH]
            [--homology HOMOLOGY] [-d DTM] [--skipdtm SKIPDTM]
            [--step STEP] [--docker DOCKER] [--ploidy PLOIDY]

Chorus2 Software for Oligo FISH probe design

optional arguments:
-h, --help            show this help message and exit
--version             show program\'s version number and exit
-j JELLYFISH, --jellyfish JELLYFISH
                      The path where Jellyfish software installed
-b BWA, --bwa BWA     The path where BWA software installed
-g GENOME, --genome GENOME
                      Fasta format genome file, should include all sequences
                      from genome
-i INPUT, --input INPUT
                      Fasta format input file, can be whole genome, a
                      chromosome or one region from genome
-s SAVED, --save SAVED
                      The output folder for saving results
-p PRIMER, --primer PRIMER
                      A specific 5\' labeled R primer for PCR reaction. For
                      example: CGTGGTCGCGTCTCA. (Default is none)
-t THREADS, --threads THREADS
                      Number of threads or CPUs to use. (Default: 1)
-l LENGTH, --length LENGTH
                      The probe length. (Default: 45)
--homology HOMOLOGY   The maximum homology(%) between target sequence and
                      probe, range from 50 to 100. (Default: 75)
-d DTM, --dtm DTM     The minimum value of dTm (hybrid Tm - hairpin Tm),
                      range from 0 to 37. (Default: 10)
--skipdtm SKIPDTM     skip calculate dtm, for oligo longer than 50.
--step STEP           The step length for k-mer searching in a sliding
                      window, step length>=1. (Default: 5)
--docker DOCKER       Only used in Docker version of Chorus
--ploidy PLOIDY       The ploidy of the given genome (test version).
                      (Default: 2)

Example:
Chorus2 -i TAIR10_chr_all.fas -g TAIR10_chr_all.fas -t 4 \
        -j /opt/software/jellyfish/bin/jellyfish -b /opt/software/bwa/bwa -s sample

Run chorus2 software

$ Chorus2 -i TAIR10_chr_all.fas -g TAIR10_chr_all.fas -t 12

When job finish, the oligo probes will output to ‘probes’ folder (Default, can be changed using -s)

$ cd sample
$ ls -lt *

    total 1741428
    -rw-r--r-- 1 root root  280927981 Aug 24 17:44 TAIR10_chr_all.fas_all.bed
    -rw-r--r-- 1 root root   62050561 Aug 24 17:44 TAIR10_chr_all.fas.bed
    -rw-r--r-- 1 root root         94 Aug 24 17:30 TAIR10_chr_all.fas.len
    -rw-r--r-- 1 root root 1031512169 Aug 24 17:22 TAIR10_chr_all.fas_tmp_probe.fa
    -rw-r--r-- 1 root root   59833928 Aug 24 17:19 TAIR10_chr_all.fas.sa
    -rw-r--r-- 1 root root       7535 Aug 24 17:18 TAIR10_chr_all.fas.amb
    -rw-r--r-- 1 root root        682 Aug 24 17:18 TAIR10_chr_all.fas.ann
    -rw-r--r-- 1 root root   29916939 Aug 24 17:18 TAIR10_chr_all.fas.pac
    -rw-r--r-- 1 root root  119667836 Aug 24 17:18 TAIR10_chr_all.fas.bwt
    -rw-r--r-- 1 root root  121183059 Aug 24 17:17 TAIR10_chr_all.fas
    -rw-r--r-- 1 root root   78102510 Aug 24 17:17 TAIR10_chr_all.fas_17mer.jf

TAIR10_chr_all.fas.bed is the probe file contained non-overlapped probes.

TAIR10_chr_all.fas_all.bed is the probe file contained all probes. This file can be used for ChorusNGSfilter.

TAIR10_chr_all.fas.len is the length info of the given genome chromosomes. This file can be imported into ChorusPBGUI for probe selection.

TAIR10_chr_all.fas_17mer.jf is the binary file created by jellyfish count using 17-mer.

TAIR10_chr_all.fas_tmp_probe.fa contains all candidate probe sequences filtered by jellyfish.

.bwt, .pac, .ann, .amb, .sa files are bwa index files.

$ more probes/TAIR10_chr_all.fas.bed
1           52      96      TCCCTAAATCTTTAAATCCTACATCCATGAATCCCTAAATACCTA
1           211     255     TTTGAGGTCAATACAAATCCTATTTCTTGTGGTTTTCTTTCCTTC
1           346     390     CCTTAGGGTTGGTTTATCTCAAGAATCTTATTAATTGTTTGGACT
1           426     470     TTTGTGGAAATGTTTGTTCTATCAATTTATCTTTTGTGGGAAAAT
1           496     540     TCTTCGTTGTTGTTACGCTTGTCATCTCATCTCTCAATGATATGG
1           551     595     TAGCATTTATTCTGAAGTTCTTCTGCTTGATGATTTTATCCTTAG

There are four columns in each row, first column is chromosome name, second is oligo start site, third is oligo end site, the last one is oligo probe sequence. You can use excel or text editor to open this file.

Further filter using ChorusNGSfilter

To further filter putative repetitive sequences, a kmer-based method can be performed to detect repeats by running ChorusNGSfilter. Before running ChorusNGSfilter, a set of whole-genome shotgun sequencing data is required. Here we download the shotgun reads of Arabidopsis with the accession number SRR5658649.

$ wget -c ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR565/009/SRR5658649/SRR5658649_1.fastq.gz
$ wget -c ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR565/009/SRR5658649/SRR5658649_2.fastq.gz

$ ChorusNGSfilter -i SRR5658649_1.fq.gz,SRR5658649_2.fq.gz -z gz \
                  -g TAIR10_chr_all.fas -t 12 \
                  -p probes/TAIR10_chr_all.fas_all.bed -o probes/TAIR10_chr_all_SRR5658649.bed

After running NGS filtering, three files (*.jf, *.bw, *.bed) will output to working directory.

TAIR10_chr_all_SRR5658649.bed.jf is the binary file created by jellyfish count using given k-mer (Default is 17).

TAIR10_chr_all_SRR5658649.bed.bw is a bigwig file contained all score infomation generated from NGS library.

TAIR10_chr_all_SRR5658649.bed is the the probe file contained all probes as well as k-mer score and strand. This file should be further selected by ChorusNGSselect.

$ more probes/TAIR10_chr_all_SRR5658649.bed
1   12      56      AAACCCTAAACCCTAAACCTCTGAATCCTTAATCCCTAAATCCCT   455128  +
1   18      62      TAAACCCTAAACCTCTGAATCCTTAATCCCTAAATCCCTAAATCT   346         +
1   24      68      CTAAACCTCTGAATCCTTAATCCCTAAATCCCTAAATCTTTAAAT   343         +
1   36      80      ATCCTTAATCCCTAAATCCCTAAATCTTTAAATCCTACATCCATG   319         +
1   42      86      AATCCCTAAATCCCTAAATCTTTAAATCCTACATCCATGAATCCC   315         +
1   48      92      TAAATCCCTAAATCTTTAAATCCTACATCCATGAATCCCTAAATA   294         +

There are six columns in each row, first four columns are the same as TAIR10_chr_all.fas_all.bed. The fifth column is the k-mer score, last column is target strand of probes.

Automatic probe selection using ChorusNGSselect

Probes should be filtered by kmer score, the process can be done by ChorusNGSselect.

$ ChorusNGSselect -i probes/TAIR10_chr_all_SRR5658649.bed \
                  -o probes/TAIR10_chr_all_SRR5658649_filter.bed

ChorusNGSselect will generate a final filtered probe file, it looks this:

$ more probes/TAIR10_chr_all_SRR5658649_filter.bed
1    36      80      ATCCTTAATCCCTAAATCCCTAAATCTTTAAATCCTACATCCATG   319     +
1    66      110     CGGGTTTAGGGAATTAGGTATTTAGGGATTCATGGATGTAGGATT   221     -
1    215     259     AGGTCAATACAAATCCTATTTCTTGTGGTTTTCTTTCCTTCACTT   293     +
1    245     289     ATAACAAATGAAGATAAACCATCCATAGCTAAGTGAAGGAAAGAA   291     -
1    347     391     CTTAGGGTTGGTTTATCTCAAGAATCTTATTAATTGTTTGGACTG   237     +
1    425     469     TTTTCCCACAAAAGATAAATTGATAGAACAAACATTTCCACAAAG   360     -

The final probes can be synthesized directly for oligo-FISH or imported into ChorusPBGUI for further selection.

Run Chorus2 with GUI

Make a project folder

$ cd ~
$ mkdir sampleproject
$ cd sampleproject

Download reference genome

$ wget https://www.arabidopsis.org/download_files/Genes/TAIR10_genome_release/TAIR10_chromosome_files/TAIR10_chr_all.fas

Run ChorusGUI

$ ChorusGUI

Set your own parameters and click Run to start the design process.

When job finish, the oligo probes will output to Sample Folder where you set.

Further filter using ChorusNGSfilter

The same process as “Run Chorus2 in terminal”

Automatic probe selection using ChorusNGSselect

The same process as “Run Chorus2 in terminal”

Run ChorusPBGUI

After filtering the probes, users can select suitable number of probes in specific regions for their FISH experiments using ChorusPBGUI easily.

$ ChorusPBGUI

Use ChorusHomo to design oligo probes for close related species

In this tutorial, we will design oligo probes for potato genome and detect conserved oligos in its close related species, tomato.

Run ChorusHomo

Make a project folder

$ cd ~
$ mkdir sampleproject
$ cd sampleproject

Download Reference Genome file

$ wget http://solanaceae.plantbiology.msu.edu/data/potato_dm_v404_all_pm_un.fasta.zip
$ unzip potato_dm_v404_all_pm_un.fasta.zip
$ wget ftp://ftp.solgenomics.net/tomato_genome/assembly/build_3.00/S_lycopersicum_chromosomes.3.00.fa

Run Chorus2 to design probes in source species

$ Chorus2 -i potato_dm_v404_all_pm_un.fasta -g potato_dm_v404_all_pm_un.fasta -t 12 -s DM404

Run ChorusHomo to design probes for close related species

This step requires more memory than Chorus2, thus set the threads smaller will be safer.

$ ChorusHomo -ga potato_dm_v404_all_pm_un.fasta -gb S_lycopersicum_chromosomes.3.00.fa \
             -i DM404/potato_dm_v404_all_pm_un.fasta_all.bed -t 8

Check the designed probes

Output files will be saved to “probes” folder. The final file potato_dm_v404_all_pm_un.fasta.homo.csv contains the conserved probes between potato and tomato genome.

The csv file contains 10 data per line:

$ head probes/potato_dm_v404_all_pm_un.fasta.homo.csv
index,probe_seq,genomeA_chr,genomeA_start,genomeA_end,genomeA_identity,genomeB_chr,genomeB_start,genomeB_end,genomeB_identity
1,ATTTTCCATGGACCTCATTAAGATTAGCTATTGAACCAGTTACCC,chr01,97858,97902,0.99,SL3.0ch07,2272165,2272211,0.70
2,ACAGCCAAATCGTCCCATATTCAAGGATAAACGACCCACGAATCA,chr01,103008,103052,0.99,SL3.0ch07,59525112,59525156,0.80
3,ATCTATATCTACTACACCAGAATATTCATACACAAATAAATTACT,chr01,127095,127139,0.99,SL3.0ch06,8431503,8431548,0.83
4,ACTGGAAGTAATATTTTGTCACAGGAATAATTCGGTAAATCTGAG,chr01,127815,127859,0.99,SL3.0ch01,27866488,27866533,0.85
5,AGGATGAAACCGTATTCCAACCTTGCTTTTCCTTTATTGATTAAT,chr01,133524,133568,0.99,SL3.0ch10,9302271,9302317,0.79
6,AAGCAGATATATCGTTCATCATACTTTATTTACATGGGGAAACAA,chr01,133859,133903,0.99,SL3.0ch10,39942300,39942345,0.78
7,TTTCTTGTGCATATTTCTAAATTGTACTGTGCAAAACTTTTCCCT,chr01,134020,134064,0.99,SL3.0ch00,85946,85991,0.83
8,AAATGATTTGCTCTTGACTGTACGTATGCCTGCCGTCTTCGTTGA,chr01,134136,134180,0.99,SL3.0ch01,63698401,63698446,0.74
9,CCTCAAGCTTACCTACAATTAGCATAGGCAGAGTTACAAGTGGAA,chr01,134190,134234,0.99,SL3.0ch01,27863901,27863944,0.91

Column 1 is index, column 2 is probe sequence, column 3-5 are probe chromosome/start/end site in genomeA (source), column 6 is the identity of probe in genomeA, column 7-9 are probe chromosome/start/end site in genomeB (target), column 10 is the identity of probe in genomeB.

Users can select high conserved probes (for example, genomeB_identity >= 0.90) for FISH experiments in close related species.

Use ChorusNoRef to design oligo probes without a reference genome

In this tutorial, we will design oligo probes for two wild potato species, S. etuberosum and S. jamesii, the two species do not have reference genomes.

Run ChorusNoRef

Make a project folder

$ cd ~
$ mkdir sampleproject
$ cd sampleproject

Download Genome file of close related species

$ wget http://solanaceae.plantbiology.msu.edu/data/potato_dm_v404_all_pm_un.fasta.zip
$ unzip potato_dm_v404_all_pm_un.fasta.zip

Download shotgun sequences of all species (at least 5x reads)

$ wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR534/006/SRR5349606/SRR5349606_1.fastq.gz
$ wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR534/006/SRR5349606/SRR5349606_2.fastq.gz
$ wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR534/003/SRR5349573/SRR5349573_1.fastq.gz
$ wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR534/003/SRR5349573/SRR5349573_2.fastq.gz
$ wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR534/004/SRR5349574/SRR5349574_1.fastq.gz
$ wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR534/004/SRR5349574/SRR5349574_2.fastq.gz

SRR5349606 is from S. tuberosum (DM404), SRR5349573 is from S. etuberosum, SRR5349574 is from S. jamesii.

Run Chorus2, ChorusNGSfilter and ChorusNGSselect to design probes in related species

Run Chorus2

$ Chorus2 -i potato_dm_v404_all_pm_un.fasta -g potato_dm_v404_all_pm_un.fasta -t 12

Run ChorusNGSfilter

$ ChorusNGSfilter -g potato_dm_v404_all_pm_un.fasta -i SRR5349606_1.fastq.gz,SRR5349606_2.fastq.gz -t 12 \
                  -p probes/potato_dm_v404_all_pm_un.fasta_all.bed -o potato_dm_v404_all_pm_un.fasta_kmer.bed

Run ChorusNGSselect

$ ChorusNGSselect -i potato_dm_v404_all_pm_un.fasta_kmer.bed -o potato_dm_v404_all_pm_un.fasta_kmerfiltered.bed

Run ChorusNoRef to design probes in target species

$ ChorusNoRef -g potato_dm_v404_all_pm_un.fasta -p potato_dm_v404_all_pm_un.fasta_kmerfiltered.bed \
              -r1 SRR5349573_1.fastq.gz,SRR5349574_1.fastq.gz -r2 SRR5349573_2.fastq.gz,SRR5349574_2.fastq.gz \
              -n etuberosum,jamesii -t 12

Check the designed probes

Output files will be saved to “noRefprobes” folder. 5 files generated.

$ ls -lh noRefprobes
-rw-rw-r-- 1 liu liu 5.2M 6月  28 17:14 etuberosum_indel_probe.txt
-rw-rw-r-- 1 liu liu 104M 6月  28 17:14 etuberosum_jamesii_cns_probe.csv
-rw-rw-r-- 1 liu liu  62M 6月  28 17:14 etuberosum_probe.txt
-rw-rw-r-- 1 liu liu 5.2M 6月  28 17:14 jamesii_indel_probe.txt
-rw-rw-r-- 1 liu liu  62M 6月  28 17:14 jamesii_probe.txt
drwxrwxr-x 2 liu liu 4.0K 6月  28 16:25 tmp

etuberosum_probe.txt and jamesii_probe.txt are probes with SNPs or identical compared to DM. etuberosum_indel_probe.txt and jamesii_indel_probe.txt are probes with indels compared to DM. etuberosum_jamesii_cns_probe.csv is consensus probes among three species after quality filter.

For probes with SNPs:

$ head -n 3 etuberosum_probe.txt
chr00   130544  130588  AGATTTTGCCCATTCTCATGACGCTTTTGTGATTTCAAAACTTTG   366     +       AGATTTAGCTCATTTTCATGGCGATTTTGTGATTTCAAGACTTTG   4
chr00   129321  129365  AATACTATTAGATGATGACTAAGAGTAATGCTAGTGTATATAAAT   262     -       CTTTATATACACTAGCATTACTCTTAGTCATCATCTAATATTATT   3
chr00   174138  174182  TTATAGTTGTCTAGGATGGAAGGGTTCTTGATTCACTGGTGTTGA   341     -       TCAACACTAGCGAATCAAGAACCCTTCCATCCTAGACAACTATAA   2

column 1-3 is location of this probe base on reference genome, column 4 is probe from reference genome, column 5 is kmer score, column 6 is strand, column 7 is probe for S. etuberosum, column 8 is how many copy can be found in etuberosum illumina reads.

For probes with indels:

$ head -n 3 etuberosum_probe.txt
chr00   298161  298205  TGATGAAGGTGAAAGTAGCATAGATCATGGGGAGTTGTTTGGATT   456     +       etuberosum      TGATGAAGGTGAAAGTAGCATAGTGCATAGATCATGGGGAGTTGTTTGGATT
chr00   298247  298291  GAATGATGAGTCAATCTGATAATTCATAGAATCAAATTTGTATGA   281     +       etuberosum      GAGTGATGAGTCAATCCATAAAGGCACCTGATAATTCATAGAATCAAATTTGTATTA
chr00   298193  298237  TCTTTAATTTACACCATAAAGTTTACTCACAAAATCCAAACAACT   495     -       etuberosum      AGTTGTTTGGATTTTGTGAAGAGAGCAGTAAACTTTATGGTGTAAATAAAAGA

column 1-3 is location of this probe based on reference genome, column 4 is probe from reference genome, column 5 is kmer score, column 6 is strand, column 7 is sample name, column 8 is probe for S. etuberosum.

For consensus probes:

$ head -n 3 etuberosum_jamesii_cns_probe.csv
chrom,start,end,refseq,etuberosum,jamesii,consensusprobe,consensusscore,consensussite,consensusdiff
chr00,130544,130588,AGATTTTGCCCATTCTCATGACGCTTTTGTGATTTCAAAACTTTG,AGATTTAGCTCATTTTCATGGCGATTTTGTGATTTCAAGACTTTG,AGATTTAACCCATTTTCATGGCGCTTTTGTAATTTCAAGACTTTG,
AGATTTAGCCCATTTTCATGGCGCTTTTGTGATTTCAAGACTTTG,0.9407407407407408,37,8
chr00,129321,129365,AATACTATTAGATGATGACTAAGAGTAATGCTAGTGTATATAAAT,CTTTATATACACTAGCATTACTCTTAGTCATCATCTAATATTATT,CTTTATATACACTAGCATTACTCTTAGTCATCATCTAATATTGCT,
CTTTATATACACTAGCATTACTCTTAGTCATCATCTAATATTAAT,0.7407407407407407,11,35

column 1-3 is location of probe based on reference genome, column 4-6 are probes in DM, etuberosum and jamesii, respectively. consensusprobe means the consensus probe among three species. consensusscore is calculated with fomula:

(probe length * number of species - number of difference) / (probe length * number of species)

Consensussite means all identical nt in cns probe. consensusdiff means how many nt different compare with cns probe.

Video Tutorials

Youtube Playlist

Playlist: https://www.youtube.com/playlist?list=PLo8q8tqFX5J27OsuKYFpd-gOtl8Qgf70X

Bilibili Playlist

Playlist: https://www.bilibili.com/video/BV1W54y1S7qS/