Abstract
During the recent years, rapid development of sequencing technologies and a competitive market has enabled researchers to perform massive sequencing projects at a reasonable cost. As the price for the actual sequencing reactions drops, enabling more samples to be sequenced, the relative price for preparing libraries gets larger and the practical laboratory work becomes complex and tedious. We present a cost-effective strategy for simplified library preparation compatible with both whole genome- and targeted sequencing experiments. An optimized enzyme composition and reaction buffer reduces the number of required clean-up steps and allows for usage of bulk enzymes which makes the whole process cheap, efficient and simple. We also present a two-tagging strategy, which allows for multiplex sequencing of targeted regions. To prove our concept, we have prepared libraries for low-pass sequencing from 100 ng DNA, performed 2-, 4- and 8-plex exome capture and a 96-plex capture of a 500 kb region. In all samples we see a high concordance (>99.4%) of SNP calls when comparing to commercially available SNP-chip platforms.
Highlights
Since the introduction of massively parallel DNA sequencing, there has been a rapid adoption of the different technologies in the sequencing field
To investigate the importance of the three variables we prepared libraries from 100 ng DNA using all combinations of the variables and performed quantitative PCR on the ligation products
An analysis of variance (ANOVA) table was constructed using the Cy0-values from the quantitative PCR (qPCR) as outcome
Summary
Since the introduction of massively parallel DNA sequencing, there has been a rapid adoption of the different technologies in the sequencing field. In order to prepare a sample for sequencing, genomic DNA is sheared and end-repaired after which common adapter sequences, often containing barcodes, are ligated onto each fragment. This step is critical as a low efficiency in the ligation step yields a low number of amplifiable DNA templates for the downstream PCR step. Automated protocols circumventing spin columns have been devised [10], capable of handling large numbers of samples. An issue with these protocols is that robotics are necessary to reach a large throughput
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