Sequencing
SFAF2016%20Meeting%20Guide%20Final%203
SFAF2016%20Meeting%20Guide%20Final%203
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11th Annual <strong>Sequencing</strong>, Finishing, and Analysis in the Future Meeting<br />
THE IMPACT OF ENZYMATIC FRAGMENTATION<br />
AND LIMITED LIBRARY AMPLIFICATION ON DATA<br />
QUALITY FOR HUMAN WHOLE-GENOME LIBRARIES<br />
SEQUENCED ON THE ILLUMINA HISEQ X<br />
Wednesday, 1st June 20:00 La Fonda NM Room (1st floor) Poster (PS‐1b.07)<br />
Maryke Appel 1 , Beverley Van Rooyen 1 , Jacqueline Meyer 1 , Penny Smorenburg 1 ,<br />
Lisa Cook 2 , Catrina Fronick 2 , Vincent Magrini 2 , Robert Fulton 2<br />
1 Roche Diagnostics, 2 Washington University<br />
Illumina’s HiSeq X platform is making the “$1,000 genome” and large‐scale human whole‐genome<br />
sequencing (WGS) a reality. Robust, high‐throughput pipelines for the production of high‐quality<br />
human genomic shotgun libraries will be needed to sustain the predicted global upsurge in human<br />
WGS research. PCR‐free library construction workflows are accepted to be the gold standard for<br />
these projects, but pose several challenges—including low and variable library yields from limited<br />
amounts of input DNA, and library stability issues.<br />
In this study we optimized library construction parameters for libraries to be sequenced on the HiSeq<br />
X; using the KAPA Hyper Prep Kit (which requires Covaris shearing), or with the KAPA HyperPlus<br />
Kit with integrated enzymatic fragmentation. Different size selection and cleanup strategies were<br />
evaluated to achieve optimal library fragment sizes (350 370 bp) and yields ( 2 nM); and minimize<br />
adapter‐dimer carry‐over.<br />
To address many of the pain points associated with high‐throughput PCR‐free workflows, we performed<br />
an amplification titration (0, 2, 4 or 6 cycles) of NA12878 libraries prepared with optimized<br />
KAPA Hyper Prep and KAPA HyperPlus protocols, and sequenced these libraries to ~30X cov‐ erage<br />
(1 library per lane equivalent). The impact of enzymatic fragmentation and low levels of PCR<br />
amplification on library QC metrics, library complexity, coverage uniformity and variant call<br />
statistics—and the implications for high‐throughput library construction pipelines for human WGS<br />
research will be discussed.<br />
Products are for life science research use only, not for use in diagnostic procedures.<br />
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