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Sample GSM1833275

Query DataSets for GSM1833275

Status

Public on Nov 04, 2015

Title

IC52

Sample type

SRA

Source name

cell-free DNA

Organism

Homo sapiens

Characteristics

tissue: blood plasma
disease: Pancreatic cancer (undefined)
Sex: M

Extracted molecule

genomic DNA

Extraction protocol

Bulk human peripheral blood plasma, containing contributions from an unknown number of healthy individuals, was obtained from STEMCELL Technologies (Vancouver, British Columbia, Canada) and stored in 2 ml aliquots at -80ºC until use. Individual human peripheral blood plasma from anonymous, healthy donors was obtained from Conversant Bio (Huntsville, Alabama, USA) and stored in 0.5 ml aliquots at -80ºC until use. Human peripheral blood plasma for 52 individuals with clinical diagnosis of Stage IV cancer was obtained from Conversant Bio or PlasmaLab International (Everett, Washington, USA) and stored in 0.5 ml or 1 ml aliquots at -80ºC until use. Human peripheral blood plasma for four individuals with clinical diagnosis of systemic lupus erythematosus was obtained from Conversant Bio and stored in 0.5 ml aliquots at -80ºC until use.
Barcoded double-strand preparation sequencing libraries were constructed with the ThruPLEX-FD or ThruPLEX DNA-seq 48D kits (Rubicon Genomics), comprising a proprietary series of end-repair, ligation, and amplification reactions. Between 0.5 ng and 30.0 ng of cfDNA were used as input for all clinical sample libraries. Library amplification for all samples was monitored by real-time PCR to avoid over-amplification, and was typically terminated after 4-6 cycles. Single-strand ligation libraries were prepared from dephosphorylated cell-free DNA. For dephosphorylation, 2X CircLigase II buffer (Epicentre), 5 mM MnCl2, and 1U FastAP alkaline phosphatase (Thermo Fisher) and 0.5-10 ng fragments were incubated in a 20 ul reaction volume at 37ºC for 30 minutes. Fragments were then denatured by heating to 95ºC for 3 minutes, and were immediately transferred to an ice bath. The reaction was supplemented with the biotin-conjugated adapter oligo (5 pmol), 20% PEG-6000 (w/v), and 200U CircLigase II (Epicentre) for a total volume of 40 ul, and was incubated overnight with rotation at 60ºC, heated to 95ºC for 3 minutes, and placed in an ice bath. For each sample, 20 ul MyOne C1 beads (Life Technologies) were twice washed in bead binding buffer (BBB) (10 mM Tris-HCl [pH 8], 1M NaCl, 1 mM EDTA [pH 8], 0.05% Tween-20, and 0.5% SDS), and resuspended in 250 ul BBB. Adapter-ligated fragments were bound to the beads by rotating for 60 minutes at room temperature. Beads were collected on a magnetic rack and the supernatant was discarded. Beads were washed once with 500 ul wash buffer A (WBA) (10 mM Tris-HCl [pH 8], 1 mM EDTA [pH 8], 0.05% Tween-20, 100 mM NaCl, 0.5% SDS) and once with 500 ul wash buffer B (WBB) (10 mM Tris-HCl [pH 8], 1 mM EDTA [pH 8], 0.05% Tween-20, 100 mM NaCl). Beads were combined with 1X Isothermal Amplification Buffer (NEB), 2.5 uM oligo CL9, 250 uM (each) dNTPs, and 24U Bst 2.0 DNA Polymerase (NEB) in a reaction volume of 50 ul, incubated with gentle shaking by ramping temperature from 15ºC to 37ºC at 1ºC/minute, and held at 37ºC for 10 minutes. After collection on a magnetic rack, beads were washed once with 200 ul WBA, resuspended in 200 ul of stringency wash buffer (SWB) (0.1X SSC, 0.1% SDS), and incubated at 45ºC for 3 minutes. Beads were again collected and washed once with 200 ul WBB. Beads were then combined with 1X CutSmart Buffer (NEB), 0.025% Tween-20, 100 uM (each) dNTPs, and 5U T4 DNA Polymerase (NEB) and incubated with gentle shaking for 30 minutes at room temperature. Beads were washed once with each of WBA, SWB, and WBB as described above. Beads were then mixed with 1X CutSmart Buffer (NEB), 5% PEG-6000, 0.025% Tween-20, 2 uM double-stranded adapter 2, and 10U T4 DNA Ligase (NEB), and incubated with gentle shaking for 2 hours at room temperature. Beads were washed once with each of WBA, SWB, and WBB as described above, and resuspended in 25 ul TET buffer (10 mM Tris-HCl [pH 8], 1 mM EDTA [pH 8], 0.05% Tween-20). Second strands were eluted from beads by heating to 95ºC, collecting beads on a magnetic rack, and transferring the supernatant to a new tube. Library amplification for all samples was monitored by real-time PCR to avoid over-amplification, and required an average of 4 to 6 cycles per library.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Description

processed data file(s): CA01.bb

Data processing

All libraries were sequenced on HiSeq 2000 or NextSeq 500 instruments (Illumina). Barcoded paired end (PE) Illumina sequencing data was split allowing up to one substitution in the barcode sequence. Reads shorter or equal to read length were consensus called and adapter trimmed.
Remaining consensus single end reads (SR) and the individual PE reads were aligned to the human reference genome sequence (GRCh37, 1000 Genomes phase 2 technical reference downloaded from ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/) using the ALN algorithm implemented in BWA v0.7.1041. PE reads were further processed with BWA SAMPE to resolve ambiguous placement of read pairs or to rescue missing alignments by a more sensitive alignment step around the location of one placed read end. Aligned SR and PE data was directly converted to sorted BAM format using the samtools API. BAM files of the sample were merged across lanes and sequencing runs.
PE provides information about the two physical ends of DNA molecules used in sequencing library preparation. We extract this information using the SAMtools application programming interface (API) from BAM files. As read starts, we use both outer alignment coordinates of PE data for which both reads aligned to the same chromosome and where reads have opposite orientations. In cases where PE data was converted to single read data by adapter trimming, we consider both end coordinates of the SR alignment as read starts. For coverage, we consider all positions between the two (inferred) molecule ends, including these end positions. We define window protection scores (WPS) of a window size k as the number of molecules spanning a window minus those starting at any bases encompassed by the window. We assign the determined WPS to the center of the window. For molecules in the 35-80 bp range (short fraction), we use a window size of 16 and, for molecules in the 120-180 bp (long fraction), we use a window size of 120.
Local maxima of nucleosome protection are called from the long fraction WPS, which we locally adjust to a running median of zero (1 kb window) and smooth using a Savitzky-Golay filter (window size 21, 2nd order polynomial). The WPS track is then segmented into above zero regions (allowing up to 5 consecutive positions below zero). If the resulting region is between 50-150 bp long, we identify the median value of that region and search for the maximum-sum contiguous window above the median. We report the start, end and center coordinates of this window. Peak-to-peak distances, etc., are calculated from the center coordinates. The score of the call is determined as the distance between maximum value in the window and the average of the two adjacent WPS minima neighboring the region. If the identified region is 150-450 bp long, we apply the same above median contiguous window approach, but only report those windows that are between 50-150bp in size. For score calculation of multiple windows derived from the 150-450bp regions, we assume the neighboring minima within the region to be zero. We discard regions shorter than 50 bp and longer than 450 bp.
Genome_build: GRCh37, 1000 Genomes phase 2 technical reference downloaded from ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/
Supplementary_files_format_and_content: Local maxima of nucleosome protection in bigBED (UCSC http://genome.ucsc.edu/FAQ/FAQformat.html#format1.5)

Submission date

Jul 27, 2015

Last update date

May 15, 2019

Contact name

Jay Shendure

Organization name

University of Washington

Department

Genome Sciences

Lab

Shendure