Browse
Submit Data
Databases
API
Help

Dataset Information

7 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Barcoded primers used in multiplex amplicon pyrosequencing bias amplification.

ABSTRACT: "Barcode-tagged" PCR primers used for multiplex amplicon sequencing generate a thus-far-overlooked amplification bias that produces variable terminal restriction fragment length polymorphism (T-RFLP) and pyrosequencing data from the same environmental DNA template. We propose a simple two-step PCR approach that increases reproducibility and consistently recovers higher genetic diversity in pyrosequencing libraries.

SUBMITTER: Berry D

PROVIDER: S-EPMC3209180 | biostudies-other | 2011 Nov

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Publications

Barcoded primers used in multiplex amplicon pyrosequencing bias amplification.

Berry David D Ben Mahfoudh Karim K Wagner Michael M Loy Alexander A

Applied and environmental microbiology 20110902 21

"Barcode-tagged" PCR primers used for multiplex amplicon sequencing generate a thus-far-overlooked amplification bias that produces variable terminal restriction fragment length polymorphism (T-RFLP) and pyrosequencing data from the same environmental DNA template. We propose a simple two-step PCR approach that increases reproducibility and consistently recovers higher genetic diversity in pyrosequencing libraries. ...[more]

PMID: 21890669

Similar Datasets

Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex.

Project description: Not available

| S-EPMC3439997 | biostudies-literature

Simulate_PCR for amplicon prediction and annotation from multiplex, degenerate primers and probes.

Project description: Not available

| S-EPMC4226945 | biostudies-literature

Epigenetic age-predictions in mice using pyrosequencing, droplet digital PCR or barcoded bisulfite amplicon sequencing

Project description:Age-associated DNA methylation reflects aspect of biological aging - therefore epigenetic clocks for mice can help to elucidate the impact of treatments or genetic background on the aging process in this model organism. Initially, age-predictors for mice were trained for genome-wide DNA methylation profiles and we have recently described a targeted assay based on pyrosequencing of DNA methylation at only three CG dinucleotides (CpGs). Here, we have re-evaluated pyrosequencing approaches in comparison to droplet digital PCR (ddPCR) and barcoded bisulfite amplicon sequencing (BBA-seq). At individual CpGs the correlation of DNA methylation with chronological age was slightly higher for pyrosequencing and ddPCR as compared to BBA-seq. On the other hand, BBA-seq revealed that neighboring CpGs tend to be stochastically modified at murine age-associated regions. Furthermore, the binary sequel of methylated and non-methylated CpGs in individual reads can be used for single-read predictions, which may reflect heterogeneity in epigenetic aging. In comparison to C57BL/6 mice the epigenetic age-predictions using BBA-seq were also accelerated in the shorter-lived DBA/2 mice, and in C57BL/6 mice with a lifespan quantitative trait locus of DBA/2 mice. Taken together, we describe further optimized and alternative targeted methods to determine epigenetic clocks in mice.

2020-08-14 | GSE156193 | GEO

Exemplary multiplex bisulfite amplicon data used to demonstrate the utility of Methpat.

Project description:DNA methylation is a complex epigenetic marker that can be analysed using a wide variety of methods. Interpretation and visualisation of DNA methylation data can mask complexity in terms of methylation status at each CpG site, cellular heterogeneity of samples and allelic DNA methylation patterns within a given DNA strand. Bisulfite sequencing is considered the gold standard, however visualisation of massively parallel sequencing results remains a significant challenge. We created a program called Methpat that facilitates visualisation and interpretation of bisulfite sequencing data generated by massively parallel sequencing. To demonstrate this, we performed multiplex PCR that targeted 48 regions of interest across 95 human samples. The regions selected included known gene promoters associated with cancer, repetitive elements, known imprinted regions and mitochondrial genomic sequences. We interrogated a range of samples including human cell lines, primary tumours and primary tissue samples. Methpat generates two forms of output: a tab delimited text file for each sample that summarises DNA methylation patterns and their read counts for each amplicon and a HTML file that summarises this data visually. Methpat can be used with publicly available whole genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS) datasets with sufficient read depths. Using Methpat, complex DNA methylation data derived from massively parallel sequencing can be summarised and visualised for biological interpretation. By accounting for allelic DNA methylation states and their abundance in a sample, Methpat can unmask the complexity of DNA methylation and reveal further biological insight in existing datasets.

2015-11-23 | GSE71804 | GEO