For questions about the scripts or C programs in this project please contact Pankaj Kumar (pankjrf@gmail.com or pk7zuva@gmail.com or pk7z@virginia.edu)

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CircularDNA_finder

A method to identify Circular DNA (Micro DNA) from pair-end high-throughput sequencing data

Cite: Kumar P, Dillon LW, Shibata Y, Jazaeri AA, Jones DR, Dutta A. Normal and Cancerous Tissues Release Extrachromosomal Circular DNA (eccDNA) into the Circulation. Mol. Cancer Res. Sep; 15(9): 1197-1205, 2017.

Cite: Laura W. Dillon, Kumar P, Shibata Y, Smaranda Willcox, Jack D. Griffith, Yves Pommier, Shunichi Takeda, Anindya Dutta. Production of extrachromosomal microDNAs is linked to mismatch repair pathways and transcriptional activity. Cell Reports; 11:1749-59, 2015.

Initially this method was described in following article

Shibata Y, Kumar P, Layer R, Willcox S, Gagan JR, Griffith JD, Dutta A. Extrachromosomal microDNAs and chromosomal microdeletions in normal tissues. Science. 2012 Apr 6;336(6077):82-6. doi: 10.1126/science.1213307. Epub 2012 Mar 8.

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Table of Contents

    Requirement
    
    **Followings are the software requirements to run the circle identification program:**

            An aligner like Bowtie (for read length less than 75) and run it without allowing soft clipping parameter
             bowtie2 (https://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.3.5.1/)
            bedtools (https://github.com/arq5x/bedtools2)
            samtools (http://samtools.sourceforge.net)
            parallel (https://www.gnu.org/software/parallel/)
                 bwa (https://github.com/lh3/bwa)
          samblaster (https://github.com/GregoryFaust/samblaster)

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    All the above tools should be installed on the system. 

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Important Note about the update:

**If your sequencing (paired-end) read length is >= 75 bases long and your sample was also enriched for circular DNA then you may use any of the two following script ** "microDNA.InOne.sh" or "circle_finder-pipeline-bwa-mem-samblaster.sh". The first one (microDNA.InOne.sh) has higher specificity but takes longer time to run.

Important Note for identifying putative circular DNA from ATAC-seq, Whole genome sequencing, Whole exome sequencing etc. Use the following script if the read length is >=75

https://github.com/pk7zuva/Circle_finder/blob/master/circle_finder-pipeline-bwa-mem-samblaster.sh

**Note: If your sample was not enriched for circular DNA (like normal ATAC-seq, whole-genome sequencing, etc.) and read length >75 bases long then use the following script ** "circle_finder-pipeline-bwa-mem-samblaster.sh"

**Note: Circle_finder can not be used if your sample was not enriched for circular DNA before library preparation AND length of read <75.

#Usage: bash Script_name "Number of processors" "/path-of-whole-genome-file/hg38.fa" "fastq file 1" "fastq file 2" "minNonOverlap between two split reads" "Sample name" "genome build"

#bash /path-of-script-dirctory/microDNA-pipeline-bwa-mem-samblaster.sh 16 /path-of-script-dirctory/hg38.fa 1E_S1_L1-L4_R1_001.fastq.75bp-R1.fastq 1E_S1_L1-L4_R2_001.fastq.75bp-R2.fastq 10 1E hg38

#Arg1 = Number of processors

#Arg2 = Genome or index file "/hdata1/MICRODNA-HG38/hg38.fa"

#Arg3 = fastq file 1 "1E_S1_L1-L4_R1_001.fastq"

#Arg4 = fastq file 2 "1E_S1_L1-L4_R2_001.fastq"

#Arg5 = minNonOverlap between two split reads "10"

#Arg6 = Sample name "1E"

#Arg7 = genome build "hg38"

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Below instruction are to run Circle_Finder if your read length is <75 bp

Usage: bash "microDNA.InOne.sh" "firstfastqfile_R1_001.fastq" "secondfastqfile_R2_001.fastq" "samplename" "Island.Mapped-Unmapped_file.Intersect_PE.bed"

Replace the samplename with what ever name you would like to give to your sample. At the end you get a list of circular DNA with number of junctional sequence. It also will give the information about presence of direct repeat at junction (this is one of the property seen with circular DNA).

Welcome to the Circle_finder wiki!

Note: Before you start the below Steps user need to install bowtie2 (https://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.3.5.1/), bedtools, samtools, parallel, bwa and samblaster (https://github.com/GregoryFaust/samblaster) in their system.

Also one need to download the whole genome and index file link provided in to "download-link-hg38-and-bowtie-index.txt" file if you would like to test the pipeline.

This is a step by step guide to run Circle_Finder (if your sample was enriched for circular DNA and the read length of your paired-end sequencing library is <75 bases).###

Step 1: Clone the repository

git clone https://github.com/pk7zuva/Circle_finder.git

Step 2: Change to "Circle_finder" directory

cd Circle_finder

In this directory you will find four types of files: 1) *.c 2) *.sh 3) *.txt and 4) C executable that has no extension

Note: Though the "C" executable files are provided it is advisable to make these executable afresh

Step 3: Type the following command on your terminal one by one

cc -o ADDRESS2PROFILEPAIREND address2profile.pairend.c

cc -o DIRECT.REPEAT.FINDER1 direct.repeat.finder1.c

cc -o JUNCTIONAL.TAG junctional.tag.c

cc -o LEFT.ALIGNMENT left.alignment.c

cc -o MIDNA_START_END_SCORE midna_start_end_score.c

Step 4: Download the whole genome files and bowtie index files from link given in file "download-link-hg38-and-bowtie-index.txt"

cat download-link-hg38-and-bowtie-index.txt

http://genome.bioch.virginia.edu/CIRCLE_FINDER_MASTER/hg38.fa http://genome.bioch.virginia.edu/CIRCLE_FINDER_MASTER/hg38.1.bt2 http://genome.bioch.virginia.edu/CIRCLE_FINDER_MASTER/hg38.2.bt2 http://genome.bioch.virginia.edu/CIRCLE_FINDER_MASTER/hg38.3.bt2 http://genome.bioch.virginia.edu/CIRCLE_FINDER_MASTER/hg38.4.bt2 http://genome.bioch.virginia.edu/CIRCLE_FINDER_MASTER/hg38.fa.amb http://genome.bioch.virginia.edu/CIRCLE_FINDER_MASTER/hg38.fa.ann http://genome.bioch.virginia.edu/CIRCLE_FINDER_MASTER/hg38.fa.bwt http://genome.bioch.virginia.edu/CIRCLE_FINDER_MASTER/hg38.fa.fai http://genome.bioch.virginia.edu/CIRCLE_FINDER_MASTER/hg38.fa.pac http://genome.bioch.virginia.edu/CIRCLE_FINDER_MASTER/hg38.fa.sa http://genome.bioch.virginia.edu/CIRCLE_FINDER_MASTER/hg38.rev.1.bt2 http://genome.bioch.virginia.edu/CIRCLE_FINDER_MASTER/hg38.rev.2.bt2

Example download command: wget http://genome.bioch.virginia.edu/CIRCLE_FINDER_MASTER/hg38.fa

Step 5: Download the fastq files. Link to download these files is given in file "fastq-file-download-link.txt"

cat fastq-file-download-link.txt

http://genome.bioch.virginia.edu/CIRCLE_FINDER_MASTER/Index11_1.fq http://genome.bioch.virginia.edu/CIRCLE_FINDER_MASTER/Index11_2.fq

Step 6: You are all set to run the pipeline

bash /path-of-the-"Circle_finder"-directory/microDNA.InOne.sh /path-of-the-"Circle_finder"-directory/hg38 Index11_1.fq Index11_2.fq 24 C4-2 49 10000 /path-of-the-"Circle_finder"-directory &

#Arg1 is bowtie2 index file "/hdata1/CIRCLE_ANALYSIS/MICRODNA-HG38/hg38"

#Arg2 is fastq file 1 "fastqfile1"

#Arg3 is fastq file 2 "fastqfile2"

#Arg4 is # of processor "24" Higher the number lower would be run time.

#Arg5 is Sample name "C4-2" in this case because circular DNA was isolated from C4-2 cell lines. You want give anyname to sample

#Arg6 Read length "49"

#Arg7 Longest circle wish to identify "10000" Higher this number more time it would take to finish the run.

#Arg8 Path of script directory

Step 7: Final output file "microDNA.JT.postmotif.fa"

head microDNA.JT.postmotif.fa chr1 28761 29551 0 1 NOMOTIF

chr1 199385 199915 0 1 GTC

chr1 631932 632604 0 1 NOMOTIF

chr1 632019 632252 1 0 CA

chr1 632112 632242 0 1 T

chr1 889483 890225 4 0 C

chr1 897103 898784 2 0 C

chr1 980217 981339 0 1 G

chr1 982484 982697 1 0 NOMOTIF

chr1 983705 984358 0 2 C

Step 8: Explanation of output

Column 1 "Chromosome name"

Column 2 "start position of circle"

Column 3 "end position of circle"

Column 4 "Number of reads mapping on circle junction from "+" strand"

Column 5 "Number of reads mapping on circle junction from "-" strand"

Column 6 "micro homology (if any) at the junction of circle"

Step 9: If you wish to extract only those circular DNA that has evidence of at least one read mapping on circle junction as "+" and "-" orientation

awk '$4>0 && $5>0' microDNA.JT.postmotif.fa | head

chr1 1069854 1070524 1 2 C

chr1 1069934 1071919 6 2 NOMOTIF

chr1 1070501 1070786 1 2 GAGTC

chr1 1428170 1428595 5 5 NOMOTIF

chr1 1459119 1460224 6 2 NOMOTIF

chr1 1459425 1462380 3 1 GGG

chr1 1495168 1495816 1 3 GG

chr1 1579383 1580962 1 1 GTA

chr1 1667878 1668245 9 6 C

chr1 1772882 1773318 2 3 A