Download Automated Targeted Locus Amplification for Targeted

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.
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