Oct 24, 2018

Fragmentation of pooled PCR products for highly multiplexed TILLING

BioRxiv : the Preprint Server for Biology
Andrea TramontanoBradley J Till

Abstract

Improvements to massively parallel sequencing have allowed the routine recovery of natural and induced sequence variants. A broad range of biological disciplines have benefited from this, ranging from plant breeding to cancer research. The need for high sequence coverage to accurately recover single nucleotide variants and small insertions and deletions limits the applicability of whole genome approaches. This is especially true in organisms with a large genome size or for applications requiring the screening of thousands of individuals, such as the reverse-genetic technique known as TILLING, where whole genome approaches become cost prohibitive. Using PCR to target and sequence chosen genomic regions provides an attractive alternative as the vast reduction in interrogated bases means that sample size can be dramatically increased through amplicon multiplexing and multi-dimensional sample pooling while maintaining suitable coverage for recovery of small mutations. Direct sequencing of PCR products is limited, however, due to limitations in read lengths of many next generation sequencers. In the present study we show the optimization and use of ultrasonication for the simultaneous fragmentation of multiplexed PCR amplicons for T...Continue Reading

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Mentioned in this Paper

Study
Size
G-protein-coupled receptor interacting protein GIP, human
Ultrasonic Surgical Procedures
Abnormal Fragmented Structure
Genome
Cancer Research
Parallel Study
Gene Deletion
Nucleic Acid Sequencing

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