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Mixed culture

ABSTRACT: Ion Torrent Bias Experiment

PROVIDER: PRJNA167265 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR515922.fastq.gz	Fastqsanger.gz
	SRR515923.fastq.gz	Fastqsanger.gz
	SRR515924.fastq.gz	Fastqsanger.gz
	SRR515925.fastq.gz	Fastqsanger.gz
	SRR515926.fastq.gz	Fastqsanger.gz

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Evaluating bias-reducing protocols for RNA sequencing library preparation

Project description:The ligation step in RNA sequencing library generation is a known source of bias. We present the first comparison of the standard duplex adaptor protocol supplied by Life Technologies for use on the Ion Torrent PGM with an alternate single adaptor approach involving CircLigase (CircLig). We also investigate whether using the thermostable ligase Methanobacterium thermoautotrophicum RNA ligase K97A (Mth K97A) for the initial ligation step in the CircLigase protocol reduces bias. A pool of small RNA fragments of known composition was converted into a sequencing library using one of three protocols and sequenced on an Ion Torrent PGM. The single adaptor CircLigase-based approach significantly reduces, but does not eliminate, bias in Ion Torrent data. Using Mth K97A as part of the CircLig method does not further reduce bias.

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Project description:The recent development of a semiconductor-based, non-optical DNA sequencing technology promises scalable, low-cost and rapid sequence data production. The technology has previously been applied mainly to genomic sequencing and targeted re-sequencing. Here, we demonstrate the utility of Ion Torrent semiconductor-based sequencing for sensitive, efficient and rapid chromatin immunoprecipitation followed by sequencing (ChIP-seq) through the application of sample preparation methods that are optimized for ChIP-seq on the Ion Torrent platform. We leverage this method for epigenetic profiling of tumor tissues.

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