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| Status |
Public on Oct 15, 2024 |
| Title |
F111501 |
| Sample type |
SRA |
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| Source name |
Tumour_CHOL
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Tumour_CHOL
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Frozen tissue sections were immersed in 420 μL of RLT Plus buffer (QIAGEN) containing tris(2-carboxyethyl)phosphine (a reducing agent; TCEP) and a unique sample tracking DNA plasmid, and gently agitated overnight at room temperature. Lysates were transferred from 2 mL tubes to wells of a 1.2 mL plate (Thermo Scientific, AB1127) to which was added 400 mL of 5x bind buffer (80 mL beads in 320 mL isopropyl alcohol). Following a 5 minute incubation at room temperature lysates were cleared on a Magnum FLX magnet place (Alpaqua Inc) for 6 minutes and the protein-containing supernatant removed. The beads, with bound nucleic acids, were washed by pipetting 10 times in wash buffer and returned to the magnet. Beads were washed three times in 70% ethanol then dried for 10 minutes. 40 mL nuclease-free water was added to the dried beads and returned to the magnet. The eluted total nucleic acids were transferred to a 96-well storage plate and aliquots taken for quantification using Invitrogen Qubit 4 Fluorometer (Thermo Fisher Q33226) For samples with concentrations below 166 ng/µL or in volumes greater than 30 µL, 5000 ng of Total Nucleic Acid (TNA) was transferred to a 1.5 mL DNase/RNase free tube and concentrated without heat on the Savant SpeedVac Plus (SC210A) to a maximum volume of 30 µL. This 5000 ng sample in 30 µL was topped up with 10 µL of Qiagen Buffer EB (Cat.19086). The samples were then run on the SAGE Science Blue Pippin instrument using the High Pass Plus Cassette (BPPLUS 10) with a maximum of 4 samples per run. The start range was selected at 15,000 base pairs and the end range was selected at 150,000 base pairs yielding a targeted size of 82,500 base pairs. Following the run completion, each sample was eluted in 80 µL and each sample elution well was then washed with 80 µL of 10 mM Tris, 1mM EDTA containing 0.1% Tween to maximize sample recovery. The total volume of 160µL was then concentrated without heat to 50 µL using the SpeedVac Plus. Each sample was then quantified using the Invitrogen Qubit 4 Fluorometer (Q33226) and normalized to 2000 ng in 47.5 µL for PromethION genome sequencing.
Library construction and sequencing followed the Oxford Nanopore Technologies Genomic DNA by Ligation (SQK-LSK110) protocol. DNA libraries starting with 2 µg of sample per library. No shearing was performed. The NEBUltra-II kit, (New England Biolabs, Ipswich, MA, USA, cat. no. E7646A) was used for end-repair and A-tailing. NEBNext quick ligase (E6056S) was used to ligate the Oxford Nanopore sequencing adapter. A final size selection of 0.4:1 ratio beads:library (PCRClean – DX Aline Biosciences L/N 06180316) was done to select against smaller molecules. These library preparation steps were performed on Hamilton Nimbus96 liquid handlers. An example deck layout, in this case for the bead purification step, is shown in Extended Data Figure 1c. DNA libraries were loaded in R9.4.1 pore flow cells on PromethION 24 instrument running software version 19.06.9 (MinKNOW GUI v4.0.23). Sequencing was carried out for 72 hours. DNase I (Invitrogen cat no. AM2222) nuclease flush was performed after 24-48 hours by reloading the flow cell with the same library mix.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
PromethION |
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| Description |
20600-OCT-3_Per-Site-Unphased-Methylation, 20600-OCT-3_Per-Read-Unphased-Methylation, 20600-OCT-3_Per-Read-Phased-Methylation-Haplotype1, 20600-OCT-3_Per-Site-Phased-Methylation-Haplotype1, 20600-OCT-3_Per-Read-Phased-Methylation-Haplotype2 and 20600-OCT-3_Per-Site-Phased-Methylation-Haplotype2
Per-Site-Unphased-Methylation, Per-Read-Unphased-Methylation, Per-Read-Phased-Methylation-Haplotype1, Per-Site-Phased-Methylation-Haplotype1, Per-Read-Phased-Methylation-Haplotype2 and Per-Site-Phased-Methylation-Haplotype2
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| Data processing |
Per-site and per-read unphased methylation data detected from nanopore sequencing raw signals using nanopolish. Phased methylation data were obtained using NanoMethPhase.
Assembly: GRCh38
Supplementary files format and content: Phased and Unphased CpG methylation data in tsv.gz format.
Library strategy: NanoMethPhase
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| Submission date |
Jun 19, 2024 |
| Last update date |
Oct 15, 2024 |
| Contact name |
Marco Marra |
| E-mail(s) |
[email protected]
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| Organization name |
Canada's Michael Smith Genome Sciences Centre
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| Street address |
570 W 7th Ave
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| City |
Vancouver |
| State/province |
BC |
| ZIP/Postal code |
V5Z 4S6 |
| Country |
Canada |
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| Platform ID |
GPL26167 |
| Series (1) |
| GSE270257 |
Long-Read Sequencing of an Advanced Cancer Cohort Resolves Rearrangements, Unravels Haplotypes, and Reveals Methylation Landscapes |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM8338263_F111501_NanoMethPhase_HP1_MethylCall.tsv.gz |
2.2 Gb |
(ftp)(http) |
TSV |
| GSM8338263_F111501_NanoMethPhase_HP1_MethylFrequency.tsv.gz |
344.4 Mb |
(ftp)(http) |
TSV |
| GSM8338263_F111501_NanoMethPhase_HP2_MethylCall.tsv.gz |
2.0 Gb |
(ftp)(http) |
TSV |
| GSM8338263_F111501_NanoMethPhase_HP2_MethylFrequency.tsv.gz |
329.0 Mb |
(ftp)(http) |
TSV |
| GSM8338263_F111501_methylation.tsv.gz |
12.6 Gb |
(ftp)(http) |
TSV |
| GSM8338263_F111501_methylation_frequency.tsv.gz |
289.6 Mb |
(ftp)(http) |
TSV |
| Raw data not provided for this record |
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