Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Sequencing random oligonucleotides using standard GA II protocol

ABSTRACT: Study on sequencing biases introduced by library preparation step, namely by ligases. Comparison between standard Illumina protocol and improved High Definition (HD) protocol. Four replicates for N21 (21 random nucleotides), one replicate for N9 (9 random nucleotides), using either standard Illumina protocol or HD protocol

ORGANISM(S): synthetic construct

SUBMITTER: Helio Pais

PROVIDER: E-GEOD-36540 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Reducing ligation bias of small RNAs in libraries for next generation sequencing.

Sorefan Karim K Pais Helio H Hall Adam E AE Kozomara Ana A Griffiths-Jones Sam S Moulton Vincent V Dalmay Tamas T

Silence 20120530 1

<h4>Background</h4>The use of nucleic acid-modifying enzymes has driven the rapid advancement in molecular biology. Understanding their function is important for modifying or improving their activity. However, functional analysis usually relies upon low-throughput experiments. Here we present a method for functional analysis of nucleic acid-modifying enzymes using next generation sequencing.<h4>Findings</h4>We demonstrate that sequencing data of libraries generated by RNA ligases can reveal nove ...[more]

PMID: 22647250

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Project description:MicroRNAs (miRNAs) regulate many genes critical for tumorigenesis. We profiled miRNAs from 11 normal breast tissues, 17 non-invasive, 151 invasive breast carcinomas, and 6 cell lines by in-house-developed barcoded Solexa sequencing. miRNAs were organized in genomic clusters representing promoter-controlled miRNA expression and sequence families representing seed-sequence-dependent miRNA-target regulation. Unsupervised clustering of samples by miRNA sequence families best reflected the clustering based on mRNA expression available for this sample set. Clustering and comparative analysis of miRNA read frequencies showed that normal breast samples were separated from most non-invasive ductal carcinoma in situ and invasive carcinomas by increased miR-21 (the most abundant miRNA in carcinomas) and multiple decreased miRNA families (including mir-98/let-7), with most miRNA changes apparent already in the non-invasive carcinomas. In addition, patients that went on to develop metastasis demonstrated increased expression of mir-423, and triple negative breast carcinomas were most distinct from other tumor subtypes due to up-regulation of the mir-17~92 cluster. However, absolute miRNA levels between normal breast and carcinomas did not reveal any significant differences. We also discovered two polymorphic nucleotide variations among the more abundant miRNAs miR-181a (T19G) and miR-185 (T16G), but we did not identify nucleotide variations expected for classical tumor suppressor function associated with miRNAs. The differentiation of tumor subtypes and prediction of metastasis based on miRNA levels is statistically possible, but is not driven by deregulation of abundant miRNAs, implicating far fewer miRNAs in tumorigenic processes than previously suggested. 21 barcoded sequencing runs, including 185 unique samples and 54 samples in replicate (6 in triplicate and the remaining in duplicate). The details of each sample can be found in Supplementary Tables S1 and S2.

Dataset Information

Sequencing random oligonucleotides using standard GA II protocol

Publications

Reducing ligation bias of small RNAs in libraries for next generation sequencing.

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