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Sample GSM1447333

Query DataSets for GSM1447333

Status

Public on Nov 17, 2014

Title

AB1 individual genomic DNA_single-end

Sample type

SRA

Source name

Testis

Organism

Homo sapiens

Characteristics

genotype: PRDM9A / PRDM9B
sequencing technique: DNA-Seq

Extracted molecule

genomic DNA

Extraction protocol

For H3K4me3 CHiP-Seq and DMC1 SSDS protocols, testicular samples were obtained frozen and were directly thawed in 1% paraformaldehyde and gently dissociated. Genomic DNA for whole genome sequencing was extracted from the testicular samples before fixation with the DNeasy Blood and Tissue kit (Qiagen).
DMC1 and H3K4Me3 ChIP were performed as described previously (Smagulova et al., 2011; Khil et al., 2012; Brick et al., 2012) with minor modifications. Sequencing libraries for anti-H3K4Me3 samples were prepared according to manufacturer’s protocol (Illumina) and anti-DMC1 libraries were prepared following the method described in (Khil et al., 2012). For genomic DNA sequencing, DNA was sheared using a Covaris S series (Covaris, MS, USA) focused-ultrasonicator. Standard sequencing library construction was done using enzymes from New England Biolabs according to the protocol provided by Illumina. The PCR amplified library was size selected on a 2% agarose gel to extract the 300 to 500 bp band.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Data processing

Basecalls performed using CASAVA version 1.8
Reads were aligned to the hg19 genome assembly. BWA was used to align genomic DNA and H3K4me3 ChIP-Seq data, while the pipeline described in Khil et al. Genome Res. 2012 was used to align SSDS reads to the genome.
For SSDS DMC1 data, hotspots were called using MACS version 2.0.10 and matched control data. The following MACS arguments were used (--nomodel; --shiftsize: 400; --bw: 1000; -q: 0.1). For the H3K4me3 ChIP-Seq experiment, SICER v1.1 (Zang et al., 2009) was used for peak calling. The following arguments were used (species: hg19; redundancy threshold: 1; window size: 200; fragment size: 200; effective genome fraction: 0.8; gap size: 200; FDR: 0.1).
Genome_build: hg19
Supplementary_files_format_and_content: For each SSDS experiment, the associated BED file contains all ssDNA fragments identified for that sample. The bed file columns are as follows: 1. Chromosome 2. Fragment start 3. Fragment end 4. Read1Quality_Read2Quality 5. ITRLength_microhomologyLength 6. Fragment strand; Peak positions called by MACS or SICER are available on the series record. The VCF file on the series record contains the variant calls.

Submission date

Jul 28, 2014

Last update date

May 15, 2019

Contact name

Kevin Brick

E-mail(s)

brickkm@mail.nih.gov, kevbrick@gmail.com, brickkm@niddk.nih.gov

Organization name

NIDDK

Department

GBB