scholarly journals Development of a robust DNA quality and quantity assessment qPCR assay for targeted next-generation sequencing library preparation

2016 ◽  
Vol 49 (4) ◽  
pp. 1755-1765 ◽  
Author(s):  
Jennifer Dang ◽  
Pedro Mendez ◽  
Sharon Lee ◽  
James W. Kim ◽  
Jun-Hee Yoon ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Qpcr Assay ◽  
Library Preparation ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Dna Quality ◽  
Sequencing Library ◽  
Sequencing Library Preparation ◽  
Generation Sequencing

scholarly journals qPCR-based characterization of DNA fragmentation efficiency of Tn5 transposomes

2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Vera Rykalina ◽  
Alexey Shadrin ◽  
Hans Lehrach ◽  
Tatiana Borodina
Keyword(s):  
Next Generation Sequencing ◽  
Dna Fragmentation ◽  
Fragment Size ◽  
Library Preparation ◽  
Next Generation ◽  
Amplification Curve ◽  
Fragmentation Efficiency ◽  
Sequencing Library ◽  
Sequencing Library Preparation ◽  
Generation Sequencing

Abstract Here, we describe an electrophoresis free assay for characterizing Tn5 transposomes fragmentation efficiency in a tagmentation reaction, in which double-stranded DNA is fragmented and tagged with adapter sequences. The assay uses plasmid DNA as a reference tagmentation substrate. Fragmentation efficiency is analyzed by comparative qPCR which measures the difference (ΔCt) in amplification of a specific plasmid region before and after tagmentation: more efficient fragmentation is characterized by a larger number of cleavage events within the amplified region, a delayed increase in the amplification curve and as a result, a larger ΔCt. Tagmentation reactions characterized with the same ΔCt exhibit the same fragment size distribution on an agarose gel. The ΔCt values measured can be used to quantitatively determine the relative performance of Tn5 transposome assemblies in optimization experiments and to standardize between batch variations in transposomes for use in next-generation sequencing library preparation. Moreover, the use of a reference tagmentation template added during next-generation sequencing library preparation enabled monitoring of the input DNA fragmentation. The presented qPCR-based assay is quick, contamination-safe, high-throughput and cost-efficient.

scholarly journals Optimizing illumina next-generation sequencing library preparation for extremely at-biased genomes

BMC Genomics ◽  
2012 ◽  
Vol 13 (1) ◽  
pp. 1 ◽  
Author(s):  
Samuel O Oyola ◽  
Thomas D Otto ◽  
Yong Gu ◽  
Gareth Maslen ◽  
Magnus Manske ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Library Preparation ◽  
Next Generation ◽  
Sequencing Library ◽  
Sequencing Library Preparation ◽  
Generation Sequencing

scholarly journals Palindromic Sequence Artifacts Generated during Next Generation Sequencing Library Preparation from Historic and Ancient DNA

PLoS ONE ◽  
2014 ◽  
Vol 9 (3) ◽  
pp. e89676 ◽  
Author(s):  
Bastiaan Star ◽  
Alexander J. Nederbragt ◽  
Marianne H. S. Hansen ◽  
Morten Skage ◽  
Gregor D. Gilfillan ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Ancient Dna ◽  
Palindromic Sequence ◽  
Library Preparation ◽  
Next Generation ◽  
Sequencing Library ◽  
Sequencing Library Preparation ◽  
Generation Sequencing
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