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A P P L I C AT I O N N O T E Automated Liquid Handling Authors: Erik Splinter Elzo de Wit Max van Min Cergentis, Utrecht The Netherlands Michiel Reessink PerkinElmer, Inc. Waltham MA Automated Targeted Locus Amplification (TLA) Technology for Targeted Complete Gene Sequencing Using the JANUS NGS Express Workstation Introduction The TLA Technology constitutes a paradigm shift in targeted next generation sequencing (NGS). The TLA technology uses the physical proximity of nucleotides within a locus of interest as the basis of selection. DNA is cross-linked, fragmented and ligated. Only one to a few primer pairs specific for a genetic locus of interest are required for the amplification of an entire locus. Any gene of interest can be amplified by TLA using a primer pair specific for the gene of interest. Generated amplicons can be processed with standard NGS library preparation technique. TLA enrichment in combination with NGS is a very flexible and easy approach to sequence regions of interest and identify all single nucleotide variants (SNVs) and structural variants (Figure 1). As such, the TLA Technology provides unique advantages in targeted sequencing of genes in comprehensive gene-fusion sequencing and in the characterization of transgenes and their integration sites. TLA sample preparation, like all NGS applications, necessitates robust library preparation to yield high-quality DNA samples. Errors in library preparation lead to loss of precious samples, wasted reagents and sequencing delays. Automated library preparation solutions like the JANUS® NGS Express Workstation shown here, do not only increase throughput and walkaway time, but also eliminate inter-operator variability in sample preparation performance as well as errors in sample tracking. Here we describe the automation of TLA Technology using the JANUS NGS Express Workstation. The JANUS NGS Express Workstation is a compact, flexible and easy to use library preparation system for benchtop sequencers such as the Ion Torrent PGM™ and MiSeq® systems. It is designed for efficient sample preparation of up to 24 samples per run. Automated sample preparation provides robust reproducible library quality, while increasing efficiency and eliminating tedious repetitive procedures (Figure 2). Materials and Methods The workflow of the TLA technology comprises these steps: 1) physical DNA crosslinking, 2) fragmentation of cross-linked DNA, 3) circularization of DNA fragments by ligation, and 4) amplification of circularized DNA with primer pair(s) specific for the genetic locus of interest (Figure 3). Figure 1. Summary of the TLA based targeted amplification and sequencing. TLA requires one primer pair complementary to a short locus specific sequence in the gene of interest for amplification. The resulting amplicons can be sequenced using Next Generation Sequencing. The complete workflow for the generation of TLA templates requires three days followed by TLA amplification and NGS library preparation on day four. The total hands-on time of the manual and automated protocols (including and excluding automated centrifugation and DNA concentration measurements) is specified in Figure 4. For this application note, the TLA protocol was performed to analyze the BRCA1 gene in the human Coriell 12878 cell-line. Starting with input material of five million viable frozen cells, 10 μg of TLA template was generated. The quality of generated template was controlled using PerkinElmer’s LabChip® GX system (Figure 5). Amplifications were performed with four primer pairs across the BRCA1 gene using 600 ng of TLA template per amplification (Figure 6). Libraries generated with these ligated fragments following Illumina’s Nextera® XT protocol yielded between 4 to 10 μg DNA (average yield: 7 μg for 24 samples). Libraries were sequenced on an Illumina MiSeq™ using mate pair sequencing with a read length of 2 x 150 bp. Figure 3. The TLA Protocol. 2 Figure 2. JANUS NGS Express Workstation. Results Figure 7 provides a sequence coverage profile generated across the BRCA1 gene and the identified SNVs across the BRCA1 gene. Mean coverage across the BRCA1 gene was 4580 and the median coverage 1863. 99% of the entire gene was covered with > 100x. Generated sequencing data was compared to the Illumina platinum genome for the same cell line. Of the 104 SNVs annotated in the full-length gene sequence 103 were confirmed. Conclusions The JANUS NGS Express Workstation offers automated sample preparation to generate high yield and quality TLA libraries providing excellent data in sequencing. The automated TLA Technology allowed rapid targeted complete sequencing of the BRCA1 gene from the human Coriell 12878 cell-line. The entire workflow and automation protocol for the generation of TLA templates and subsequent target locus amplification is identical for the amplification of any gene(s) of interest, only a different set of target-specific TLA primers will be used. These amplifications can also be multiplexed. As such, the automation of TLA Technology permits the fast, robust and highly flexible targeted sequencing of any combination of genes of interest. Figure 4. Hands-on time of the manual protocol, current automated with and without automated centrifugation and DNA concentration measurement. Figure 5. LabChip® GX electropherogram run profile of ligated BRCA1 TLA product (1:5 dilution). Figure 6. The positions of the four primer pairs used for the targeted amplification of the BRCA1 gene. Figure 7. Generated sequence coverage across the BRCA1 gene. Colored bars represent the position of hetero/ homozygous SNVs with respect to the reference genome. For research use only. Not for use in Diagnostic procedures. PerkinElmer, Inc. 940 Winter Street Waltham, MA 02451 USA P: (800) 762-4000 or (+1) 203-925-4602 www.perkinelmer.com For a complete listing of our global offices, visit www.perkinelmer.com/ContactUs Copyright ©2014-2016, PerkinElmer, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners. 011756A_01