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DeepSAGE--digital transcriptomics with high sensitivity, simple experimental protocol and multiplexing of samples.

ABSTRACT: Digital transcriptomics with pyrophosphatase based ultra-high throughput DNA sequencing of di-tags provides high sensitivity and cost-effective gene expression profiling. Sample preparation and handling are greatly simplified compared to Serial Analysis of Gene Expression (SAGE). We compare DeepSAGE and LongSAGE data and demonstrate greater power of detection and multiplexing of samples derived from potato. The transcript analysis revealed a great abundance of up-regulated potato transcripts associated with stress in dormant potatoes compared to harvest. Importantly, many transcripts were detected that cannot be matched to known genes, but is likely to be part of the abiotic stress-response in potato.

SUBMITTER: Nielsen KL

PROVIDER: S-EPMC1636492 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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DeepSAGE--digital transcriptomics with high sensitivity, simple experimental protocol and multiplexing of samples.

Nielsen Kåre L KL Høgh Annabeth Laursen AL Emmersen Jeppe J

Nucleic acids research 20061005 19

Digital transcriptomics with pyrophosphatase based ultra-high throughput DNA sequencing of di-tags provides high sensitivity and cost-effective gene expression profiling. Sample preparation and handling are greatly simplified compared to Serial Analysis of Gene Expression (SAGE). We compare DeepSAGE and LongSAGE data and demonstrate greater power of detection and multiplexing of samples derived from potato. The transcript analysis revealed a great abundance of up-regulated potato transcripts ass ...[more]

PMID: 17028099

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Project description:ChIP-seq experiments are standard experimental procedure for interrogating epigenetic states and protein-DNA interactions. Sequencing experiments are often designed according to the trade-off between the need to obtain maximum sequencing coverage limited funds. Multiplexing samples is a common approach to minimize cost and maximize information yield. We therefore performed an extensive ChiP-seq multiplexing study to gain a better understanding of the effect of multiplexing on the resulting peak detection and genomic annotation and to provide solid guidelines for multiplexing ChIP-seq studies. For a well characterized antibody, our results indicate that multiplexing to ~20M reads (roughly 8 samples per sequencing lane) is sufficient to capture most of the biological signal. Multiplexing samples in sequencing experiments is a common approach to maximize information yield while minimizing cost. In most cases the number of samples that are multiplexed is determined by financial consideration or experimental convenience with limited understanding on the effects on the experimental results. Here we set to examine the impact of multiplexing ChIP-seq experiments on the ability to identify a specific epigenetic modification. We performed an analysis of peak detection to determine the effects of multiplexing. These include false discovery rates, size, position and statistical significance of peak detection and changes in gene annotation. We found that, for histone marker H3K4me3, one can multiplex up to 8 samples (7 IP + 1 input) at ~21 million reads each and still detect over 90% of all peaks found when using a full lane for sample. Furthermore, there are no variations introduced by indexing or lane batch effects and importantly there is no significant reduction in the number of genes with neighboring H3K4me3 peaks. We conclude that, for a well characterized antibody and therefore, model IP condition, multiplexing 8 samples per lane is sufficient to capture most of the biological signal.

Dataset Information

DeepSAGE--digital transcriptomics with high sensitivity, simple experimental protocol and multiplexing of samples.

Publications

DeepSAGE--digital transcriptomics with high sensitivity, simple experimental protocol and multiplexing of samples.

Similar Datasets

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