Agilent NGS Automation
Citation Index

Agilent NGS Automation

In this Citation Index, you’ll gain fast access to the bibliographic information you
need to research emerging data, review trends and stay up-to-date with the latest
Agilent NGS Automation application being performed by genomic researchers
around the world. It contains a variety of research areas that are utilizing the valueof automation to increase throughput and maintain consistent data quality.
Cost-effective, high-throughput DNA sequencing
libraries for multiplexed targetcapture
Nadin Rohland, David Reich
Genome Research, Volume 22, Pages 939-946, 2012

Improvements in technology have reduced the cost of
DNA sequencing to the point that the limiting factor
for many experiments is the time and reagent cost of
sample preparation. We present an approach in which 192
sequencing libraries can be produced in a single day oftechnician time at a cost of about $15 per sample. These
libraries are effective not only for low-pass whole-genome
sequencing, but also for simultaneously enriching them inpools of approximately 100 individually barcoded samples fora subset of the genome without substantial loss in efficiencyof target capture. We illustrate the power and effectiveness of
this approach on about 2000 samples from a prostate cancer
study.

Pre-capture multiplexing improves efficiency andcost-effectiveness of targeted genomic enrichment
A Eliot Shearer, Michael S Hildebrand, Harini Ravi, Swati Joshi,
Angelica C Guiffre, Barbara Novak, Scott Happe, Emily M LeProust,
Richard JH Smith
BMC Genomics, Volume 13, Page 618, 2012

Targeted genomic enrichment (TGE) is a widely usedmethod for isolating and enriching specific genomic regions
prior to massively parallel sequencing. To make effective
use of sequencer output, barcoding and sample pooling(multiplexing) after TGE and prior to sequencing (post-capture
multiplexing) has become routine. While previous reports
have indicated that multiplexing prior to capture (pre-capture
multiplexing) is feasible, no thorough examination of theeffect of this method has been completed on a large numberof samples. Here we compare standard post-capture TGE
to two levels of pre-capture multiplexing: 12 or 16 samples
per pool. We evaluated these methods using standard TGE
metrics and determined the ability to identify several classes

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of genetic mutations in three sets of 96 samples, including
48 controls. Our overall goal was to maximize cost reduction
and minimize experimental time while maintaining a highpercentage of reads on target and a high depth of coverage atthresholds required for variant detection.

A High-Throughput Chromatin Immunoprecipitation
Approach Reveals Principles of Dynamic Gene
Regulation in Mammals
Manuel Garber, Nir Yosef, Alon Goren, Raktima Raychowdhury, Anne
Thielke, Mitchell Guttman, James Robinson, Brian Minie, Nicolas
Chevrier, Zohar Itzhaki, Ronnie Blecher-Gonen, Chamutal Bornstein,
Daniela Amann-Zalcenstein, Assaf Weiner, Dennis Friedrich, James
Meldrim, Oren Ram, Christine Cheng, Andreas Gnirke, Sheila Fisher,
Nir Friedman, Bang Wong, Bradley E. Bernstein, Chad Nusbaum, Nir
Hacohen, Aviv Regev, Ido Amit
Molecular Cell, Volume 47, Pages 810-822, 2012

Understanding the principles governing mammalian gene
regulation has been hampered by the difficulty in measuringin vivo binding dynamics of large numbers of transcription
factors (TF) to DNA. Here, we develop a high-throughput
Chromatin ImmunoPrecipitation (HT-ChIP) method tosystematically map protein-DNA interactions. HT-ChIP
was applied to define the dynamics of DNA binding by
25 TFs and 4 chromatin marks at 4 time-points following
pathogen stimulus of dendritic cells. Analyzing over 180,000
TF-DNA interactions we find that TFs vary substantially intheir temporal binding landscapes. This data suggests a
model for transcription regulation whereby TF networks
are hierarchically organized into cell differentiation factors,
factors that bind targets prior to stimulus to prime them forinduction, and factors that regulate specific gene programs.
Overlaying HT-ChIP data on gene-expression dynamics
shows that many TF-DNA interactions are established prior
to the stimuli, predominantly at immediate-early genes, andidentified specific TF ensembles that coordinately regulate
gene-induction.

A scalable, fully automated process for constructionof sequence-ready human exome targeted capture
libraries
Sheila Fisher, Andrew Barry, Justin Abreu, Brian Minie, Jillian Nolan,
Toni M Delorey, Geneva Young, Timothy J Fennell, Alexander Allen,
Lauren Ambrogio, Aaron M Berlin, Brendan Blumenstiel, Kristian
Cibulskis, Dennis Friedrich, Ryan Johnson, Frank Juhn, Brian Reilly,
Ramy Shammas, John Stalker, Sean M Sykes, Jon Thompson, John
Walsh, Andrew Zimmer, Zac Zwirko, Stacey Gabriel, Robert Nicol andChad Nusbaum
Genome Biology, Volume 12, Page R1, 2011

Genome targeting methods enable cost-effective captureof specific subsets of the genome for sequencing. We
present here an automated, highly scalable method forcarrying out the Solution Hybrid Selection capture approachthat provides a dramatic increase in scale and throughput
of sequence‑readylibraries produced. Significant process
improvements and a series of in-process quality control
checkpoints are also added. These process improvements
can also be used in a manual version of the protocol.

AHT-ChIP-seq: a completely automatedrobotic protocol for high-throughput chromatin
immunoprecipitation
Sarah Aldridge, Stephen Watt, Michael A Quail, Tim Rayner, Margus
Lukk, Michael F Bimson, Daniel Gaffney and Duncan T Odom
Genome Biology, Volume 14, Page R124, 2013

ChIP-seq is an established manually-performed method for
identifying DNA-protein interactions genome-wide. Here, we
describe a protocol for automated high-throughput (AHT)
ChIP-seq. To demonstrate the quality of data obtained using
AHT-ChIP-seq, we applied it to five proteins in mouse livers
using a single 96-well plate, demonstrating an extremely
high degree of qualitative and quantitative reproducibility
among biological and technical replicates. We estimated the
optimum and minimum recommended cell numbers required
to perform AHT-ChIP-seq by running an additional plate using
HepG2 and MCF7 cells. With this protocol, commerciallyavailable robotics can perform four hundred experiments infive days.

The Genetic Landscape of Clinical Resistance to
RAF Inhibition in Metastatic Melanoma
Eliezer M. Van Allen, Nikhil Wagle, Antje Sucker, Daniel J. Treacy,
Cory M. Johannessen, Eva M. Goetz, Chelsea S. Place, Amaro
Taylor‑Weiner, Steven Whittaker, Gregory V. Kryukov, Eran Hodis,
Mara Rosenberg, Aaron McKenna, Kristian Cibulskis, Deborah
Farlow, Lisa Zimmer, Uwe Hillen, Ralf Gutzmer, Simone M. Goldinger,
Selma Ugurel, Helen J. Gogas, Friederike Egberts, Carola Berking,
Uwe Trefzer, Carmen Loquai, Benjamin Weide, Jessica C. Hassel,
Stacey B. Gabriel, Scott L. Carter, Gad Getz, Levi A. Garraway and Dirk
Schadendorf
Cancer Discovery, Volume 4, Page 94-109, 2014

Most patients with BRAF(V600)-mutant metastatic melanoma
develop resistance to selective RAF kinase inhibitors. The
spectrum of clinical genetic resistance mechanisms to RAF
inhibitors and options for salvage therapy are incompletely
understood. We performed whole-exome sequencing on
formalin-fixed, paraffin-embedded tumors from 45 patientswith BRAF(V600)-mutant metastatic melanoma who received
vemurafenib or dabrafenib monotherapy. Genetic alterationsin known or putative RAF inhibitor resistancegenes wereobserved in 23 of 45 patients (51%). Besides previously
characterized alterations, we discovered a “long tail” of new
mitogen-activated protein kinase (MAPK) pathway alterations(MAP2K2, MITF) that confer RAF inhibitor resistance. In threecases, multiple resistance gene alterations were observed
within the same tumor biopsy. Overall, RAF inhibitor therapy
leads to diverse clinical geneticresistance mechanisms,
mostly involving MAPK pathway reactivation. Noveltherapeutic combinations may be needed to achieve durable
clinical control of BRAF(V600)-mutant melanoma. Integrating
clinicalgenomics with preclinical screens may model
subsequent resistance studies.

Automation of PacBio SMRTbell NGS library
preparation for bacterial genome sequencing
Nguyet Kong, Whitney Ng, Kao Thao, Regina Agulto, Allison Weis,
Kristi Spittle Kim, Jonas Korlach, Luke Hickey, Lenore Kelly, Stephen
Lappin and Bart C. Weimer
Standards in Genomic Sciences, Volume 12, Page 27, 2017

BACKGROUND: The PacBio RS II provides for single molecule,real-time DNA technology to sequence genomes and detect
DNA modifications. The starting point for high-quality
sequence production is high molecular weight genomic DNA.
To automate the library preparation process, there must be
high-throughput methods in place to assess the genomic
DNA, to ensure the size and amounts of the sheared DNA
fragments and final library.

3

FINDINGS: The library construction automation was
accomplished using the Agilent NGS workstation with Bravo
accessories for heating, shaking, cooling, and magnetic
bead manipulations for template purification. The quality
control methods from gDNA input to final library using theAgilent Bioanalyzer System and Agilent TapeStation Systemwere evaluated.
CONCLUSIONS: Automated protocols of PacBio 10 kb
library preparation produced libraries with similar technicalperformance to those generated manually. The TapeStation
System proved to be a reliable method that could be used in a
96-well plate format to QC the DNA equivalent to the standard
Bioanalyzer System results. The DNA Integrity Number that is
calculated in the TapeStation System software upon analysis
of genomic DNA is quite helpful to assure that the starting
genomic DNA is not degraded. In this respect, the gDNA
assay on the TapeStation System is preferable to the DNA
12000 assay on the Bioanalyzer System, which cannot run
genomic DNA, nor can the Bioanalyzer work directly from the
96-well plates.

Efficient method to optimize antibodies using avian
leukosis virus display and eukaryotic cells
Changming Yua, Gennett M. Pikea, Tommy A. Rinkoskia, Cristina
Correiab, Scott H. Kaufmannb, and Mark J. Federspiel
Proceedings of the National Academy of Sciences of the UnitedStates of America, Volume 112, Pages 9860-9865, 2015

Antibody-based therapeutics have now had success in theclinic. The affinity and specificity of the antibody for thetarget ligand determines the specificity of therapeutic delivery
and off-target side effects. The discovery and optimizationof high-affinity antibodies to important therapeutic targets
could be significantly improved by the availability of a robust,eukaryotic display technology comparable to phage displaythat would overcome the protein translation limitationsof microorganisms. The use of eukaryotic cells would
improve the diversity of the displayed antibodies that canbe screened and optimized as well as more seamlesslytransition into a large-scale mammalian expression systemfor clinical production. In this study, we demonstrate thatthe replication and polypeptide display characteristics ofa eukaryotic retrovirus, avianleukosis virus (ALV), offersa robust, eukaryotic version of bacteriophage display.

4

The binding affinity of a model single-chain Fv antibody
was optimized by using ALV display, improving affinity>2,000‑fold, from micromolar to picomolar levels. We believe
ALV display provides an extension to antibody display on
microorganisms and offers virus and cell display platforms
in a eukaryotic expression system. ALV displayshould enable
an improvement in the diversity of properly processed
and functional antibody variants that can be screened andaffinity‑optimized to improve promising antibody candidates.

Development of a high-resolution NGS-based
HLA‑typing and analysis pipeline
Michael Wittig, Jarl A. Anmarkrud, Jan C. Kässens, Simon Koch,
Michael Forster, Eva Ellinghaus, Johannes R. Hov, Sascha Sauer,
Manfred Schimmler, Malte Ziemann, Siegfried Görg, Frank Jacob,
Tom H. Karlsen, Andre Franke
Nucleic Acid Research, Volume 43, Page e70, 2015

The human leukocyte antigen (HLA) complex contains themost polymorphic genes in the human genome. The classical
HLA class I and II genes define the specificity of adaptive
immune responses. Genetic variation at the HLA genes is
associated with susceptibility to autoimmune and infectious
diseases and plays a major role in transplantation medicine
and immunology. Currently, the HLA genes are characterizedusing Sanger- or next-generation sequencing (NGS) of alimited amplicon repertoire or labeled oligonucleotides forallele-specific sequences. High-quality NGS-based methods
are in proprietary use and not publicly available. Here, we
introduce the first highly automated open-kit/open-source
HLA-typing method for NGS. The method employs in-solution
targeted capturing of the classical class I (HLA-A, HLA-B,
HLA-C) and class II HLA genes (HLA-DRB1, HLA-DQA1,
HLA‑DQB1, HLA-DPA1, HLA-DPB1). The calling algorithm
allows for highly confident allele-calling to three-field
resolution (cDNA nucleotide variants). The method wasvalidated on 357 commercially available DNA samples withknown HLA alleles obtained by classical typing. Our results
showed on average an accurate allele call rate of 0.99 ina fully automated manner, identifying also errors in thereference data. Finally, our method provides the flexibility to
add further enrichment target regions.

Genomic Comparison of Campylobacter spp. andTheir Potential for Zoonotic Transmission between
Birds, Primates, and Livestock
Allison M. Weis, Dylan B. Storey, Conor C. Taff, Andrea K. Townsend,
Bihua C. Huang, Nguyet T. Kong, Kristin A. Clothier, Abigail Spinner,
Barbara A. Byrne, and Bart C. Weimer
Applied and Environmental Microbiology, Volume 82,
Pages 7165-7175, 2016

Campylobacter is the leading cause of human gastroenteritis
worldwide. Wild birds, including American crows, areabundant in urban, suburban, and agricultural settings andare likely zoonotic vectors of Campylobacter Their proximity
to humans and livestock increases the potential spreadingof Campylobacter via crows between the environment,
livestock, and humans. However, no studies have definitively
demonstrated that crows are a vector for pathogenic
Campylobacter We used genomics to evaluate the zoonotic
and pathogenic potential of Campylobacter from crows
to other animals with 184 isolates obtained from crows,
chickens, cows, sheep, goats, humans, and nonhumanprimates. Whole-genome analysis uncovered two distinct
clades of Campylobacter jejuni genotypes; the first contained
genotypes found only in crows, while a second genotype
contained "generalist" genomes that were isolated frommultiple host species, including isolates implicated in humandisease, primate gastroenteritis, and livestock abortion.
Two major β-lactamase genes were observed frequently
in these genomes (oxa-184, 55%, and oxa-61, 29%), where
oxa-184 was associated only with crows and oxa-61 was
associated with generalists. Mutations in gyrA, indicativeof fluoroquinolone resistance, were observed in 14% of
the isolates. Tetracycline resistance (tetO) was present in
22% of the isolates, yet it occurred in 91% of the abortion
isolates. Virulence genes were distributed throughout the
genomes; however, cdtC alleles recapitulated the crow-onlyand generalist clades. A specific cdtC allele was associatedwith abortion in livestock and was concomitant with tetO
These findings indicate that crows harboring a generalist
C. jejuni genotype may act as a vector for the zoonotic
transmission of Campylobacter IMPORTANCE: This study
examined the link between public health and the genomic
variation of Campylobacter in relation to disease in humans,
primates, and livestock. Use of large-scale whole-genome
sequencing enabled population-level assessment to find
new genes that are linked to livestock disease. With 184
Campylobactergenomes, we assessed virulence traits,
antibiotic resistance susceptibility, and the potential forzoonotictransfer to observe that there is a “generalist”
genotype that may move between host species.

Spatial reconstruction of single-cellgene expression
Rahul Satija, Jeffrey A. Farrell, David Gennert, Alexander F. Schier, andAviv Regev
Nature Biotechnology, Volume 33, Pages 495-502, 2015

Spatial localization is a key determinant of cellularfate and behavior, but methods for spatially resolved,
transcriptome-wide gene expression profiling across complex
tissues are lacking. RNA staining methods assay only asmall number of transcripts, whereas single-cell RNA-seq,
which measures global gene expression, separates cells
from their native spatial context. Here we present Seurat,
a computational strategy to infer cellular localization
by integrating single-cell RNA-seq data with in situ RNA
patterns. We applied Seurat to spatially map 851 single
cells from dissociated zebrafish (Danio rerio) embryos andgenerated a transcriptome-wide map of spatial patterning.
We confirmed Seurat's accuracy using several experimental
approaches, then used the strategy to identify a set ofarchetypal expression patterns and spatial markers. Seurat
correctly localizes rare subpopulations, accurately mapping
both spatially restricted and scattered groups. Seurat will beapplicable to mapping cellular localization within complex
patterned tissues in diverse systems.

Massively parallel single cell RNA-Seq formarker‑free decomposition of tissues into cell types
Diego Adhemar Jaitin, Ephraim Kenigsberg, Hadas Keren-Shaul,
Naama Elefant, Franziska Paul, Irina Zaretsky, Alexander Mildner,
Nadav Cohen, Steffen Jung, Amos Tanay, and Ido Amit
Science, Volume 343, Pages 776-779, 2014

In multicellular organisms, biological function emerges whenheterogeneous cell types form complex organs. Nevertheless,dissection of tissues into mixtures of cellular subpopulations
is currently challenging. We introduce an automatedmassively parallel single-cell RNA sequencing (RNA-seq)
approach for analyzing in vivo transcriptional states inthousands of single cells. Combined with unsupervisedclassification algorithms, this facilitates ab initio cell-typecharacterization of splenic tissues. Modeling single-celltranscriptional states in dendritic cells and additional
hematopoietic cell types uncovers rich cell-type heterogeneityand gene-modules activity in steady state and after pathogen
activation. Cellular diversity is thereby approached throughinference of variable and dynamic pathway activity rather
than a fixed preprogrammed cell-type hierarchy. These data
demonstrate single-cell RNA-seq as an effective tool forcomprehensive cellular decomposition of complex tissues.

5Full-length RNA-seq from single cells using
Smart‑seq2.
Simone Picelli, Omid R Faridani, Åsa K Björklund, Gösta Winberg,
Sven Sagasser, and Rickard Sandberg
Nature Protocols, Volume 9, Pages 171-181, 2014

Emerging methods for the accurate quantification of gene
expression in individual cells hold promise for revealing theextent, function and origins of cell-to-cell variability. Different
high-throughput methods for single-cell RNA-seq have been
introduced that vary in coverage, sensitivity and multiplexing
ability. We recently introduced Smart-seq for transcriptome
analysis from single cells, and we subsequently optimized
the method for improved sensitivity, accuracy and full-length
coverage across transcripts. Here we present a detailed
protocol for Smart-seq2 that allows the generation offull‑length cDNA and sequencing libraries by using standard
reagents. The entire protocol takes ~2 d from cell picking
to having a final library ready for sequencing; sequencing
will require an additional 1-3 d depending on the strategy
and sequencer. The current limitations are the lack of strand
specificity and the inability to detect nonpolyadenylated(polyA(-)) RNA.

Detection of Low Frequency Multi-Drug Resistanceand Novel Putative Maribavir Resistance in
Immunocompromised Pediatric Patients with
CytomegalovirusCharlotte J. Houldcroft, Josephine M. Bryant, Daniel P. Depledge, Ben
K. Margetts, Jacob Simmonds, Stephanos Nicolaou, Helena J. Tutill,
Rachel Williams, Austen J. J. Worth, Stephen D. Marks, Paul Veys,
Elizabeth Whittaker, and Judith Breuer, the PATHSEEK Consortium
Frontiers in Microbiology, Volume 7, Page 1317, 2016

Human cytomegalovirus (HCMV) is a significant pathogen
in immunocompromised individuals, with the potential tocause fatal pneumonitis and colitis, as well as increasing therisk of organ rejection in transplant patients. With the adventof new anti-HCMV drugs there is therefore considerable
interest in using virus sequence data to monitor emerging
resistance to antiviral drugs in HCMV viraemia and disease,including the identification of putative new mutations. We
used target-enrichment to deep sequence HCMV DNA from
11 immunosuppressed pediatric patients receiving single
or combination anti-HCMV treatment, serially sampledover 1-27 weeks. Changes in consensus sequence andresistancemutations were analyzed for three ORFs targetedby anti-HCMV drugs and the frequencies of drug resistancemutations monitored. Targeted-enriched sequencing of

6clinical material detected mutations occurring at frequencies
of 2%. Seven patients showed no evidence of drug resistancemutations. Four patients developed drug resistancemutations a mean of 16 weeks after starting treatment. In
two patients, multiple resistance mutations accumulated at
frequencies of 20% or less, including putative maribavir andganciclovir resistance mutations P522Q (UL54) and C480F(UL97). In one patient, resistance was detected 14 days earlier
than by PCR. Phylogenetic analysis suggested recombinationor superinfection in one patient. Deep sequencing of HCMV
enriched from clinical samples excluded resistance in 7 of
11 subjects and identified resistance mutations earlier thanconventional PCR-based resistance testing in 2 patients.
Detection of multiple low level resistance mutations was
associated with poor outcome.

Elucidation of MRAS-mediated Noonan syndrome
with cardiac hypertrophy
Erin M. Higgins, J. Martijn Bos, Heather Mason-Suares, David J.
Tester, Jaeger P. Ackerman, Calum A. MacRae, Katia Sol-Church,
Karen W. Gripp, Raul Urrutia, and Michael J. Ackerman
JCI Insight, Volume 2, Page e91225, 2017

Noonan syndrome (NS; MIM 163950) is an autosomal
dominant disorder and a member of a family ofdevelopmental disorders termed "RASopathies," which
are caused mainly by gain-of-function mutations ingenes encoding RAS/MAPK signaling pathway proteins.
Whole exome sequencing (WES) and trio-based genomic
triangulation of a 15-year-old female with a clinicaldiagnosis of NS and concomitant cardiachypertrophyand her unaffected parents identified a de novo variant
in MRAS‑encoded RAS-related protein 3 as the cause ofher disease. Mutation analysis using in silico mutation
prediction tools and molecular dynamics simulations
predicted the identified variant, p.Gly23Val-MRAS, to bedamaging to normal protein function and adversely affecteffector interaction regions and the GTP-binding site.
Subsequent ectopic expression experiments revealed a40-fold increase in MRAS activation for p.Gly23Val-MRAS
compared with WT-MRAS. Additional biochemical assays
demonstrated enhanced activation of both RAS/MAPK
pathway signaling and downstream gene expression incells expressing p.Gly23Val‑MRAS. Mutational analysis
of MRAS in a cohort of 109 unrelated patients withphenotype‑positive/genotype‑negative NS and cardiac
hypertrophy yielded another patient with a sporadic de novo
MRAS variant (p.Thr68Ile, c.203C>T). Herein, we describe
the discovery of mutations in MRAS in patients with NS andcardiac hypertrophy, establishing MRAS as the newest NS
with cardiac hypertrophy-susceptibility gene.

Mapping the Hallmarks of Lung Adenocarcinoma
with Massively Parallel Sequencing
Marcin Imielinski, Alice H. Berger, Peter S. Hammerman, Bryan
Hernandez, Trevor J. Pugh, Eran Hodis, Jeonghee Cho, JamesSuh, Marzia Capelletti, Andrey Sivachenko, Carrie Sougnez, Daniel
Auclair, Michael S. Lawrence, Petar Stojanov, Kristian Cibulskis,
Kyusam Choi, Luc de Waal, Tanaz Sharifnia, Angela Brooks, Heidi
Greulich, Shantanu Banerji, Thomas Zander, Danila Seidel, Frauke
Leenders, Sascha Ansén, Corinna Ludwig, Walburga Engel-Riedel,Erich Stoelben, Jürgen Wolf, Chandra Goparju, Kristin Thompson,
Wendy Winckler, David Kwiatkowski, Bruce E. Johnson, Pasi A. Jänne,Vincent A. Miller, William Pao, William D. Travis, Harvey I. Pass, Stacey
B. Gabriel, Eric S. Lander, Roman K. Thomas, Levi A. Garraway, Gad
Getz, Matthew Meyerson
Cell, Volume 150, Pages 1107-1120, 2012

Lung adenocarcinoma, the most common subtype of
non-small cell lung cancer, is responsible for more than
500,000 deaths per year worldwide. Here, we report exome
and genome sequences of 183 lungadenocarcinoma
tumor/normal DNA pairs. These analyses revealed a mean
exonic somatic mutation rate of 12.0 events/megabaseand identified the majority of genes previously reported as
significantly mutated in lung adenocarcinoma. In addition,
we identified statistically recurrent somatic mutations inthe splicing factor gene U2AF1 and truncating mutations
affecting RBM10 and ARID1A. Analysis of nucleotide contextspecific mutation signatures grouped the sample set into
distinct clusters that correlated with smoking history andalterations of reported lung adenocarcinoma genes. Wholegenome sequence analysis revealed frequent structural
rearrangements, including in-frame exonic alterations within
EGFR and SIK2 kinases. The candidate genes identified in thisstudy are attractive targets for biological characterization andtherapeutic targeting of lung adenocarcinoma.

Whole exome analysis identifies frequent CNGA1
mutations in Japanese population with autosomal
recessive retinitis pigmentosa

METHODS:
In total, 99 Japanese patients with non-syndromic andunrelated arRP or sporadic RP (spRP) were recruited in thisstudy and ophthalmic examinations were conducted forthe diagnosis of RP. Among these patients, whole exome
sequencing analysis of 30 RP patients and direct sequencing
screening of all CNGA1 exons of the other 69 RP patientswere performed.
RESULTS:
Whole exome sequencing of 30 arRP/spRP patients identified
disease-causing gene mutationsof CNGA1 (four patients),
EYS (three patients) and SAG (one patient) in eight patientsand potential disease-causing gene variants of USH2A (twopatients), EYS (one patient), TULP1 (one patient) and C2orf71(one patient) in five patients. Screening of an additional 69
arRP/spRP patients for the CNGA1gene mutation revealed
one patient with a homozygous mutation.
CONCLUSIONS:This is the first identification of CNGA1 mutations in arRP
Japanese patients. The frequency of CNGA1 gene mutation
was 5.1% (5/99 patients). CNGA1 mutations are one of themost frequent arRP-causing mutations in Japanese patients.

Determining the frequency of pathogenic germline
variants from exome sequencing in patients withcastrate-resistant prostate cancer
Steven N Hart, Marissa S Ellingson, Kim Schahl, Peter T Vedell,
Rachel E Carlson, Jason P Sinnwell, Poulami Barman, Hugues
Sicotte, Jeanette E Eckel-Passow, Liguo Wang, Krishna R Kalari, Rui
Qin, Teresa M Kruisselbrink, Rafael E Jimenez, Alan H Bryce, Winston
Tan, Richard Weinshilboum, Liewei Wang, Manish Kohli
BMJ Open, Volume 6, Page e010332, 2016

OBJECTIVES:
To determine the frequency of pathogenic inherited mutationsin 157 select genes from patients with metastatic castrateresistant prostate cancer (mCRPC).

Satoshi Katagiri, Masakazu Akahori, Yuri Sergeev, Kazutoshi
Yoshitake, Kazuho Ikeo, Masaaki Furuno, Takaaki Hayashi, Mineo
Kondo, Shinji Ueno, Kazushige Tsunoda, Kei Shinoda, Kazuki
Kuniyoshi, Yohinori Tsurusaki, Naomichi Matsumoto, Hiroshi
Tsuneoka, Takeshi Iwata

DESIGN:

PLoS One, Volume 9, Page e108721, 2014

PARTICIPANTS:OBJECTIVE:

Seventy-one adult male patients with histologicalconfirmation of prostate cancer, and had progressive disease
while on androgen deprivation therapy.

The purpose of this study was to investigate frequentdisease-causing gene mutations in autosomal recessive
retinitis pigmentosa (arRP) in the Japanese population.

Observational.
SETTING:
Multisite US-based cohort.

7

RESULTS:
Twelve patients (17.4%) showed evidence of carrying
pathogenic or likely pathogenic germlinevariants in theATM, ATR, BRCA2, FANCL, MSR1, MUTYH, RB1, TSHR andWRN genes. All but one patient opted in to receive clinicallyactionable results at the time of study initiation. We also
found that pathogenic germline BRCA2 variants appear to beenriched in mCRPC compared to familial prostatecancers.
CONCLUSIONS:
Pathogenic variants in cancer-susceptibility genes are
frequently observed in patientswith mCRPC. A substantial
proportion of patients with mCRPC or their family members
would derive clinical utility from mutation screening.

Prognostic interaction between ASXL1 and TET2
mutations in chronic myelomonocytic leukemia
M M Patnaik, T L Lasho, P Vijayvargiya, C M Finke, C A Hanson,
R P Ketterling, N Gangat, and A Tefferi
Blood Cancer Journal, Volume 6, Page e385, 2016

Mutations involving epigenetic regulators (TET2~60% and
ASXL1~40%) and splicing components (SRSF2~50%) arefrequent in chronic myelomonocytic leukemia (CMML).
On a 27-gene targeted capture panel performed on 175
CMML patients (66% males, median age 70 years), common
mutations included: TET2 46%, ASXL1 47%, SRSF2 45%
and SETBP1 19%. A total of 172 (98%) patients had at least
one mutation, 21 (12%) had 2, 24 (14%) had 3 and 30 (17%)
had >3 mutations. In a univariate analysis, the presenceof ASXL1 mutations (P=0.02) and the absence of TET2
mutations (P=0.03), adversely impacted survival; while thenumber of concurrent mutations had no impact (P=0.3). Ina multivariable analysis that included hemoglobin, platelet
count, absolute monocyte count and circulating immature
myeloid cells (Mayo model), the presence of ASXL1 mutations(P=0.01) and absence of TET2 mutations (P=0.003) retained
prognostic significance. Patients were stratified into four
categories: ASXL1wt/TET2wt (n=56), ASXL1mut/TET2wt(n=31), ASXL1mut/TET2mut (n=50) and ASXL1wt/TET2mut(n=38). Survival data demonstrated a significant differencein favor of ASXL1wt/TET2mut (38 months; P=0.016),
compared with those with ASXL1wt/TET2wt (19 months),
ASXL1mut/TET2wt (21 months) and ASXL1mut/TET2mut(16 months) (P=0.3). We confirm the negative prognostic
impact imparted by ASXL1 mutations and suggest afavorable impact from TET2 mutations in the absence of
ASXL1 mutations.

8

Targeted deep sequencing in primary myelofibrosis
Ayalew Tefferi, Terra L. Lasho, Christy M. Finke, Yoseph Elala, Curtis
A. Hanson, Rhett P. Ketterling, Naseema Gangat, and Animesh
Pardanani
Blood Advances, Volume 1, Pages 105-111, 2016

A myeloid neoplasm–relevant 27-gene panel was used fornext-generation sequencing of bone marrow or whole blood
DNA in 182 patients with primary myelofibrosis (PMF). DNA
sequence variants/mutations other than JAK2/CALR/MPL
were detected in 147 patients (81%), with the most frequent
being ASXL1 (36%), TET2 (18%), SRSF2 (18%), and U2AF1(16%); furthermore, 35%, 26%, 10%, and 9% of the patients
harbored 1, 2, 3, or 4 or more such variants/mutations,
respectively. Adverse variants/mutations were identified
by age-adjusted multivariable analysis of impact on overall
survival or leukemia-free survival and included ASXL1,
SRSF2, CBL, KIT, RUNX1, SH2B3, and CEBPA; their combined
prevalence was 56%. Adverse variants/mutations wereassociated with inferior overall survival (median, 3.6 vs 8.5
years; P < .001) and leukemia-free survival (7-year risk, 25%
vs 4%; P < .001), and the effect on survival was independentof both the Dynamic International Prognostic Scoring
System Plus and JAK2/CALR/MPL mutational status, withrespective hazard ratios of 2.0 (95% confidence interval[CI], 1.3-3.1) and 2.9 (95% CI, 1.9-4.4). Additional prognostic
information was obtained by considering the number ofadverse variants/mutations; median survivals in patientswith zero (n = 80), 1 or 2 (n = 93), or 3 or more (n = 9) adverse
variants/mutations were 8.5, 4, and 0.7 years, respectively(P < .001). Additional data were obtained on pattern ofmutation co-segregation and phenotypic correlation, including
significant associations between U2AF1 and JAK2 mutations(P = .04) and U2AF1 mutations and anemia (P = .003)
and thrombocytopenia (P = .006). We conclude that DNA
variants/mutations other than JAK2/CALR/MPL are prevalentin PMF and are qualitatively and quantitatively relevant inpredicting overall and leukemia-free survival.

Comprehensive Assessment of Genetic Variants
Within TCF4 in Fuchs’ Endothelial Corneal Dystrophy

Hereditary hemorrhagic telangiectasia: genetics
and molecular diagnostics in a new era

Eric D. Wieben; Ross A. Aleff; Bruce W. Eckloff; Elizabeth J. Atkinson;
Saurabh Baheti; Sumit Middha; William L. Brown; Sanjay V. Patel;
Jean-Pierre A. Kocher; Keith H. Baratz

Jamie McDonald, Whitney Wooderchak-Donahue,
Chad VanSant Webb, Kevin Whitehead, David A. Stevenson, andPinar Bayrak‑Toydemir

Investigative Ophthalmology & Visual Science, Volume 55,
Pages 6101-6107, 2014

Frontiers in Genetics, Volume 6, Page 1, 2015

PURPOSE:The single nucleotide variant (SNV), rs613872, in thetranscription factor 4 (TCF4) gene was previously found to bestrongly associated (P = 6 × 10(-26)) with Fuchs’ endothelial
corneal dystrophy(FECD). Subsequently, an intronic expansionof the repeating trinucleotides, TGC, was found to be evenmore predictive of disease. We performed comprehensive
sequencing of the TCF4 gene region in order to identify thebest marker for FECD within TCF4 and to identify other novel
variants that may be associated with FECD.
METHODS:
Leukocyte DNA was isolated from 68 subjects with FECD
and 16 unaffected individuals. A custom capture panel wasused to isolate the region surrounding the two previously
validated markers of FECD. Sequencing of the TCF4 coding
region, introns and flanking sequence, spanning 465 kb was
performed at >1000× average coverage using the Illumina
HiSequation 2000.
RESULTS:TGC expansion (>50 repeats) was present in 46 (68%)
FECD‑affected subjects and one (6%) normal subject. A totalof 1866 variants, including 1540 SNVs, were identified. Only
two previously reported SNVs resided in the TCF4 coding
region, neither of which segregated with disease. No variant,
including TGC expansion, correlated perfectly with diseasestatus. Trinucleotide repeat expansion was a better predictor
of disease than any other variant.
CONCLUSIONS:
Complete sequencing of the TCF4 genomic region revealed
no single causative variant for FECD. The intronic trinucleotide
repeat expansion within TCF4 continues to be more strongly
associated with FECD than any other genetic variant.

Hereditary hemorrhagic telangiectasia (HHT) is a vasculardysplasia characterized by telangiectases and arteriovenous
malformations (AVMs) in particular locations described inconsensus clinical diagnostic criteria published in 2000.
Two genes in the transforming growth factor-beta (TGF-β)signaling pathway, ENG and ACVRL1, were discovered almost
two decades ago, and mutations in these genes have beenreported to cause up to 85% of HHT. In our experience,
approximately 96% of individuals with HHT have a mutation inthese two genes, when published (Curaçao) diagnostic criteria
for HHT are strictly applied. More recently, two additional
genes in the same pathway, SMAD4 and GDF2, have been
identified in a much smaller number of patients with asimilar or overlapping phenotype to HHT. Yet families still
exist with compelling evidence of a hereditary telangiectasia
disorder, but no identifiable mutation in a known gene. Recent
availability of whole exome and genome testing has created
new opportunities to facilitate gene discovery, identify genetic
modifiers to explain clinical variability, and potentially define
an increased spectrum of hereditary telangiectasia disorders.
An expanded approach to molecular diagnostics for inherited
telangiectasia disorders that incorporates a multi-gene next
generation sequencing (NGS) HHT panel is proposed.

BMP9 Mutations Cause a Vascular-Anomaly
Syndrome with Phenotypic Overlap with Hereditary
Hemorrhagic Telangiectasia
Whitney L. Wooderchak-Donahue, Jamie McDonald, Brendan
O’Fallon, Paul D. Upton, Wei Li, Beth L. Roman, Sarah Young, Parker
Plant, Gyula T. Fülöp, Carmen Langa, Nicholas W. Morrell, Luisa M.
Botella, Carmelo Bernabeu, David A. Stevenson, James R. Runo, Pinar
Bayrak-Toydemir
American Journal of Human Genetics, Volume 93, Pages 530-537,
2013

Hereditary hemorrhagic telangiectasia (HHT), the mostcommon inherited vascular disorder, is caused by mutations
in genes involved in the transforming growth factor beta
(TGF-β) signaling pathway (ENG, ACVRL1, and SMAD4). Yet,approximately 15% of individuals with clinical features of
HHT do not have mutations in these genes, suggesting that
there are undiscovered mutations in other genes for HHT andpossibly vascular disorders with overlapping phenotypes.

9

The genetic etiology for 191 unrelated individuals clinicallysuspected to have HHT was investigated with the use ofexome and Sanger sequencing; these individuals had no
mutations in ENG, ACVRL1, and SMAD4. Mutations in BMP9
(also known as GDF2) were identified in three unrelated
probands. These three individuals had epistaxis and dermal
lesions that were described as telangiectases but whose
location and appearance resembled lesions described insome individuals with RASA1-related disorders (capillarymalformation-arteriovenous malformation syndrome).
Analyses of the variant proteins suggested that mutations
negatively affect protein processing and/or function, and abmp9-deficient zebrafish model demonstrated that BMP9 isinvolved in angiogenesis. These data confirm a genetic causeof a vascular-anomaly syndrome that has phenotypicoverlap
with HHT.

Genetic Variants Associated with Port-Wine Stains
Alice Frigerio, Karol Wright, Whitney Wooderchak-Donahue, Oon T.
Tan, Rebecca Margraf, David A. Stevenson, J. Fredrik Grimmer,
Pinar Bayrak-Toydemir
PLoS One, Volume 10, Page e0133158, 2015

BACKGROUND:
Port-wine stains (PWS) are capillary malformations, typicallylocated in the dermis of the head and neck, affecting 0.3%of the population. Current theories suggest that port-wine
stains are caused by somatic mutations that disrupt vasculardevelopment.
OBJECTIVES:
Understanding PWS genetic determinants could provide
insight into new treatments.
METHODS:
Our study used a custom next generation sequencing (NGS)
panel and digital polymerase chain reaction to investigate
genetic variants in 12 individuals with isolated port-wine
stains. Importantly, affected and healthy skin tissue from thesame individual were compared. A subtractive correction
method was developed to eliminate background noise fromNGS data. This allowed the detection of a very low level ofmosaicism.
RESULTS:
A novel somatic variant GNAQ, c.547C>G, p.Arg183Gly was
found in one case with 4% allele frequency. The previously
reported GNAQ c.548G>A, p.Arg183Gln was confirmed in
9 of 12 cases with an allele frequency ranging from 1.73 to
7.42%. Digital polymerase chain reaction confirmed novel
variantsdetected by next generation sequencing. Two novel
somatic variants were also found in RASA1, although neither
was predicted to be deleterious.
10

CONCLUSIONS:This is the second largest study on isolated, non-syndromic
PWS. Our data suggest that GNAQ is the main genetic
determinant in this condition. Moreover, isolated port-wine
stains are distinct from capillary malformations seen in
RASA1 disorders, which will be helpful in clinical evaluation.

RASA1 somatic mutation and variable expressivity
in capillary malformation/arteriovenous
malformation (CM/AVM) syndrome
Colleen F. Macmurdo, Whitney Wooderchak-Donahue, Pinar BayrakToydemir, Jenny Le, Matthew B. Wallenstein, Carlos Milla, Joyce M. C.
Teng, Jonathan A. Bernstein, David A. Stevenson
American Journal of Medical Genetics, Volume 170,
Pages 1450‑1454, 2016

Germline mutations in RASA1 are associated with capillarymalformation-arteriovenous malformation (CM-AVM)
syndrome. CM-AVM syndrome is characterized by multi-focal
capillary malformations and arteriovenous malformations.
Lymphatic anomalies have been proposed as part of thephenotype. Intrafamilial variability has been reported,suggesting modifiers and somatic events. The objective of
the study was to identify somatic RASA1 "second hits" fromvascular malformations associated with CM-AVMsyndrome,and describe phenotypic variability. Participants were
examined and phenotyped. Genomic DNA was extracted
from peripheral blood on all participants. Whole-exome
sequencing was performed on the proband. Using Sanger
sequencing, RASA1 exon 8 was PCR-amplified to track thec.1248T>G, p.Tyr416X germline variant through the family. A
skin biopsy of a capillary malformation from the proband's
mother was also obtained, and next-generation sequencing
was performed on DNA from the affected tissue. A familial
germline heterozygous novel pathogenic RASA1 variant,c.1248T>G (p.Tyr416X), was identified in the proband andher mother. The proband had capillary malformations,chylothorax, lymphedema, and overgrowth, while her affectedmother had only isolated capillary malformations. Sequence
analysis of DNA extracted from a skin biopsy of a capillarymalformation of the affected mother showed a second
RASA1 somatic mutation (c.2245C>T, p.Arg749X). These
results and the extreme variable expressivity support the
hypothesis that somatic "second hits" are required for thedevelopment of vascular anomalies associated with CM‑AVM
syndrome. In addition, the phenotypes of the affectedindividuals further clarify that lymphatic manifestations
are also part of the phenotypic spectrum of RASA1-relateddisorders.

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