GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM4025999

Query DataSets for GSM4025999

Status

Public on Jul 20, 2020

Title

NC2

Sample type

SRA

Source name

NC2

Organism

blank sample

Characteristics

set: negative control (no template DNA)

Extracted molecule

genomic DNA

Extraction protocol

Fish were euthanized with tricaine methanesulfonate and tissues were dissected out and placed in 95% EtOH. DNA was extracted by proteinase K digestion and phenol:chloroform purification. 500 ng genomic DNA was bisulfite treated using Zymo Research EZ Methylation-Gold kit.
Four regions were amplified (Bioline Immomix) from bisulfite-treated gDNA samples using primers with Illumina-compatible tails. Fragments were cleaned (Aline Biosciences PCRCLEAN DX) before a second round of PCR to attach sample-specific barcodes. Samples were pooled at equal concentrations and column purified to produce Illumina libraries.
DNA amplicon sequencing

Library strategy

Bisulfite-Seq

Library source

genomic

Library selection

RANDOM

Instrument model

Illumina MiSeq

Description

NC2_S62_L001

Data processing

Sequence reads were aligned to in silico bisulfite converted amplicon reference sequences (https://github.com/rose-driscoll/methylation-analysis/tree/master/reference_sequences/Bisulfite_Genome/CT_conversion) with bowtie2 using options -N 1 --dovetail -x
Samtools view -bS, samtools sort, and samtools mpileup -d 200000 were used to produce a vcf file for each sample
Custom python scripts available at https://github.com/rose-driscoll/methylation-analysis/blob/master/percent-methylation/methylation_pipeline_samtoolspythonandcat_20180605_RD.txt were employed to count the number of As, Cs, Ts, and Gs observed at each base, extract data for selected bases (CpG Cs, Ts and non-CpG Cs, and As and Gs, relative to the reference sequence), and calculate relevant statistics such as % methylation for CpG C bases and % error for A and G bases.
In a second pipeline, forward and reverse reads from each sample were combined using FLASH and aligned to in silico bisulfite converted amplicon reference sequences (https://github.com/rose-driscoll/methylation-analysis/tree/master/reference_sequences/Bisulfite_Genome/CT_conversion) with bowtie2 using options -N 1 -x in order to identify which amplicon they had come from
Reads from each of the four amplicons were extracted from the bowtie alignment, dereplicated with usearch -derep_fulllength -sizeout, and aligned with muscle along with a reference sequence.
Custom python scripts available at https://github.com/rose-driscoll/methylation-analysis/tree/master/epialleles were employed to identify CpG sites in the aligned reads.
Command line tools (cut and awk) were employed to paste together CpG bases and identify an 'epiallele' for each read, and count the number of times each unique epiallele appeared for each sample.
Supplementary_files_format_and_content: tab-delimited text files with % methylation per CpG site (all_CpGs_20180605_RD.txt), bisulfite conversion completeness per base (all_conversion_20180605_RD.txt), sequencing error per base (all_error_20180605_RD.txt), or epiallele abundance (all_haplotypes_w_AP6_20180612.tab) per sample.

Submission date

Aug 10, 2019

Last update date

Aug 09, 2020

Contact name

Suzy CP Renn

Organization name

Reed College

Department

Biology

Street address

3203 SE Woodstock Blvd