 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Feb 19, 2017 |
| Title |
NC-P-62 |
| Sample type |
SRA |
| |
|
| Source name |
colorectal cancer_plasma
|
| Organism |
Homo sapiens |
| Characteristics |
disease state: normal
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Plasma from patients were processed to extract circulating DNA. The DNA extracted from plasma were then concentrated using ethanol precipitation and eluted in 15 uL nuclease-free water.
1-1.5 ng of DNA were digested with 10 U of MspI (Thermoscientific), 1X Tango buffer (Thermoscientific), and 10 pg of unmethylated lambda DNA (New England Biolabs) as control for ~13 h at 37 ˚C, then heat inactivated at 65 ˚C for 20 min. Next, 5 U of Klenow fragment, exo- (Thermoscientifc) and a mixture of dATP, dGTP, and dCTP (New England Biolabs) were added for a final concentration of 1 mM, 0.1 mM, and 0.1 mM for dATP, dGTP, and dCTP respectively. The mixture was gently vortexed, and incubated at 30 ˚C for 20 min, 37 ˚C for 20 min, and finally 75 ˚C for 10 min. To perform adaptor ligation, the dA-tailed DNA were added to a 5 uL mixture of 1X Tango buffer, 30 U of T4 DNA Ligase, HC (Thermoscientific), 2.5 mM ATP, and 500 nM individual TruSeq multiplexing methylated adaptors. The combined mixture was gently vortexed, incubated at 16 ˚C for ~20 h, then heat inactivated at 65 ˚C for 20 min. The ligation mixture was purified using Agencourt AMPure XP beads (Beckman Coulter), and eluted in 20 uL of nuclease-free water. The ligated products were then bisulfite converted using the MethylCode(™) Bisulfite Conversion kit (Life Technologies). Two rounds of amplification were performed after bisulfite conversion. The first round was using PfuTurboCX (Agilent Technologies) for 12 cycles in 50 uL total volume, then the second round was performed using Phusion HotStart Flex (New England Biolabs) master mix for 9 cycles in 50 uL total volume. Final PCR products were purified, pooled in equimolar ratios, and size selected using polyacrylamide gels for 150-400 bp. Libraries were sequenced on both MiSeq and HiSeq RapidRun for PE 100 cycles.
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Sample 156
|
| Data processing |
base-calling
quality trimming of fastq reads
alignment with BWA/bisReadMapper
trim overlapping pair-ends
geneerate methylation haplotype counts from BAM
Genome_build: hg19
Supplementary_files_format_and_content: tab separated values: column 1 - genomic locus, column 2 - methylation haplotype call, column 3 - number of reads supporting methylation haplotype, column 4 - list of CpG positions included in methylation haplotype
|
| |
|
| Submission date |
Jan 21, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Kun Zhang |
| E-mail(s) |
k4zhang@ucsd.edu
|
| Organization name |
University of California, San Diego
|
| Department |
Bioengineering
|
| Lab |
Integrative Genomics Laboratory
|
| Street address |
9500 Gilman Dr Mailcode: 0412
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE79277 |
Identification of methylation haplotype blocks aids in deconvolution of heterogeneous tissue samples and tissue-of-origin mapping from plasma DNA [Plasma RRBS] |
| GSE79279 |
Identification of methylation haplotype blocks aids in deconvolution of heterogeneous tissue samples and tissue-of-origin mapping from plasma DNA |
|
| Relations |
| BioSample |
SAMN06249425 |
| SRA |
SRX2510563 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2464841_RRBS_2016.NC-P-62.mhbs.haploInfo.bed.gz |
4.7 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
