Dataset Information

Ena-DATASET-CRUKCI-05-03-2016-10:48:49:023-8 - samples

ABSTRACT: Shallow whole-genome sequencing of samples from the study "Methanol-based fixation is superior to buffered formalin for next-generation sequencing of DNA from clinical cancer samples". DNA from each sample (100ng) was sheared on Covaris S220 (Covaris): duty cycle - 10%, intensity -5.0, bursts per sec - 200, duration - 300 sec, mode - frequency sweeping, power - 23V, temperature -5:5 C to 6 C, water level - 13. Libraries were prepared with the TruSeq Nano DNA LT Sample Prep Kit (Illumina) using a modi?ed protocol - Sample Puri?cation Beads were replaced by Agencourt AMPure XP beads (Beckman Coultier) and size selection after the End Repair was done to remove only the short fragments. Quality and quantity for contructed libraries were assessed with DNA 7500 kit on Agilent 2100 Bioanalyzer and with Kapa Quanti?cation kit (KAPA Biosystems) on 7900HT Fast Real-Time PCR System (Applied Biosystems) according to the supplier's recommendations, respectively. Libraries from 18 barcoded samples were pooled together in equimolar amounts and each pool was loaded on a single lane of a HiSeq Single End Flowcell (Illumina), followed by cluster generation on a cBot (Illumina) and sequencing on a HiSeq 2500 (Illumina) in a single-read 50bp mode. Reads were aligned using bwa-mem v0.7.12-r1039 to the 1000 genomes version of human genome build GRCh37. Picard (http://picard.sourceforge.net) was used to remove duplicate reads.

PROVIDER: EGAD00001001938 | EGA |

REPOSITORIES: EGA

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Publications

Methanol-based fixation is superior to buffered formalin for next-generation sequencing of DNA from clinical cancer samples.

Piskorz A M AM Ennis D D Macintyre G G Goranova T E TE Eldridge M M Segui-Gracia N N Valganon M M Hoyle A A Orange C C Moore L L Jimenez-Linan M M Millan D D McNeish I A IA Brenton J D JD

Annals of oncology : official journal of the European Society for Medical Oncology 20151217 3

<h4>Background</h4>Next-generation sequencing (NGS) of tumour samples is a critical component of personalised cancer treatment, but it requires high-quality DNA samples. Routine neutral-buffered formalin (NBF) fixation has detrimental effects on nucleic acids, causing low yields, as well as fragmentation and DNA base changes, leading to significant artefacts.<h4>Patients and methods</h4>We have carried out a detailed comparison of DNA quality from matched samples isolated from high-grade serous ...[more]

PMID: 26681675

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Project description:Purpose: To investigate the role of DNA methylation in human uterine leiomyoma tumorigenesis, we profiled genome-wide DNA methylation patterns of leiomyoma cells (LM) and adjacent normal myometrial cells (MM) treated with or without DNA methylation inhibitor 5-Aza. Methods: Primary cells were isolated from fresh uterine myometrium or leiomyoma tissue. Cells were treated with vehicle (DMSO) or 5 µM 5’-aza for 96h with medium refreshed every 24h. Genomic DNA was extracted from LM and MM cells using DNeasy Blood and Tissue Kit (Qiagen, 69504) and fragmented to 300-500bp using Covaris M220. Methylated DNA fragments were captured using the MethylCap Kit (Diagenode, C02020010). Next-generation sequencing libraries were prepared using the KAPA Hyper Prep Kit (KAPA Biosystems, KK8502) and KAPA Single-Indexed Adapter Kit (KAPA Biosystems, KK8710). Libraries were sequenced under 75bp paired-end sequencing format. Sequences were aligned to the hg19 reference genome using Bowtie2. Histone style peak calling, differential binding analysis (getDifferentialPeaks) and motif analysis were performed using Homer under default settings. Differentily methylated regions (DMRs) were detected with a fold enrichment over background tag count ≥4 and poisson enrichment p-value over background tag count <0.0001. Differential DNA methylation between MM and LM (with or without treatment) at select regions were validated through qPCR. Results: More than 100,000 methyalted regions were detected in each sample. We discovered 21,086 DMRs between MM and LM cells under basal (vehicle treated) condition. In cells treated with 5-aza, we detected 8811 DMRs between MM and LM. We compared the genomic context of DMRs identified by MethylCap-Seq with the probe distribution of the HM450 array and detected large differences between these two methods. For example, 44.23% of DMR were located in intergenic regions by MethylCap-Seq, which was drastically higher than the percentage of HM450 array probes located in intergenic regions Downstream network enrichment analysis and motif analysis indicated that DMR-associated genes and regions are crucial for the tumor progression and hormone responsiveness of LM. Conclusions: Our study represents the detailed analysis of differential DNA methyaltion profiles between MM and LM using MethylCap-seq. Our results showed a more comprehensive evaluation of DNA methyaltion landscapes of MM and LM compared with previous array based analysis. We conclude that DNA methylation plays a crucial role in hormone-dependent LM tumorigenesis.

Dataset Information

Ena-DATASET-CRUKCI-05-03-2016-10:48:49:023-8 - samples

Publications

Methanol-based fixation is superior to buffered formalin for next-generation sequencing of DNA from clinical cancer samples.

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