Next-generation sequencing gene panels enable the examination of multiple genes, identifying previously described variants and discovering novel variants, to elucidate genetic disease. The challenges are substantial, including: identification of all genes of interest; assay optimization to create robust, reproducible, multiplex panels; and developing accurate, comprehensive, reproducible analysis pipelines.

1. Thermo Fisher Scientific • 5781 Van Allen Way • Carlsbad, CA 92008 • thermofisher.com
Fiona Hyland, Mani Manivannan, Brijesh Krishnaswami, Yun Zhu, Yuan Tian, Heinz Breu, Cristina Van Loy, Adam Broomer, Tanya Biorac, Dominique DeWolf, Yutao Fu, Mark Andersen,,
Steve Roman. Thermo Fisher Scientific, 180 Oyster Point Blvd, South San Francisco, CA , USA and Carlsbad, CA, USA.
Creating custom gene panels for next-generation sequencing: optimization of 5000 gene assays, selection by disease
research area and integrated analysis for variant prioritization.
INTRODUCTION
Introduction: Next-generation sequencing gene panels enable the examination of multiple genes, identifying previously described variants and
discovering novel variants, to elucidate genetic disease. The challenges are substantial, including: identification of all genes of interest; assay
optimization to create robust, reproducible, multiplex panels; and developing accurate, comprehensive, reproducible analysis pipelines.
Methods: We describe a comprehensive set of tools to simplify targeted gene selection, sequencing and analysis. First we developed the
Content Selection Engine to describe the relationship between genes and diseases, enabling browsing through a disease hierarchy and
selection of genes by disease categories, along with a scoring algorithm (rank-weighted sum score, RWSS) which scores every gene by clinical
relevance for all diseases across the disease hierarchy. We optimized assays for each of 5000 disease research genes, creating a database of
empirical amplicon performance and pairing this with improved assay design tools. A cloud-based web interface enables easy selection of any
diseases, shows all associated genes, and enables selection of any associated genes and addition of more genes. We incorporate IGV to
facilitate visualization of empirical base and exon coverage. A custom Ion AmpliSeq On-Demand panel is created using the performance-
verified assays from these genes.
Smaller gene panels which are narrowly targeted to specific diseases, or broader gene panels with hundreds of genes for extended disease
categories, can be created. Genetic disease research areas include neonatal phenotypes such as metabolic disorders, Severe Combined
Immunodeficiency, musculoskeletal disorders, heme disorders; and late onset phenotypes such as cardiovascular disorders and cancer
predisposition.
Run
Sequence
Prepare
Library
Sample
Input
AnalyzePrepare Template
Oncomine™ Immune
Response Research
Assay
(Manual or
automated library
preparation)
FFPE derived total
RNA
Analysis solution on
Torrent SuiteTM
Ion S5™
System
(520 or 530)
or
Ion PGM™
System
(318)
Ion Chef™ System
or
Ion OneTouch™
2
System
Content Selection Engine
The synthesis of MeSH disease hierarchies with DisGeNET gene-disease relations forms our Content Selection Engine. MeSH provides a
mathematical graph indicating disease parent/child relationships; e.g., “autoimmune disease” is a parent disease of “rheumatoid arthritis”, while
“juvenile rheumatoid arthritis” is a child disease of “rheumatoid arthritis”.
The nodes on the graph, the diseases themselves, are related to genes through a score provided by DisGeNET for each gene-disease pair.
Taken together, the disease hierarchy combined with the gene-disease scores provide a way to look up genes involved with any disease or group
of diseases at any level in the disease hierarchy. The Gene Scoring Algorithm (GSA) summarizes child diseases’ gene contributions to a selected
parent disease in the hierarchy.
The database contains over X associations between Y genes and Z diseases, both Mendelian and complex.
METHODS
RESULTS
CONCLUSIONS
Panel based Uniformity on dried blood spots,
frozen samples (examples from 2 panels)
Robust, high performing AmpliSeq On-Demand Gene Panels
AmpliSeq On Demand assays are designed to comprehensively cover exons. While the minimum
exon padding is 5 bases, typically genes have amplicons extending 50 bases or more past the edge
of the exon. Number of amplicons per gene ranges between 1 and 303.
The optimizaton process resulted in an optimized set of gene assays which have high performance:
most of the genes have 100% in silico coverage and 100% uniformity. Uniformity is defined as the %
of bases on target having read depth ≥ 0.2X of the mean read depth. Uniformity is typically defined
per panel, and can also be measured per gene.
Disease Gene Scoring
Rank-Weighted Sum Score (RWSS) is an unbiased gene scoring method that
accounts for both the strength and number of gene-disease pairs.
The GSA module takes the DisGeNET scores as input and uses RWSS method to
prioritize genes for a specific disease of interest.
In conjunction with the Content Selection Engine, the GSA module can produce a list of
ranked genes for one or more diseases at any level of the disease hierarchy.
The DisGeNET score was developed to rank the gene-disease associations according to
their level of evidence. DisGeNET gene-disease association score takes into account the
number and type of sources (level of curation, model organisms), and the number of
publications supporting the association. The score ranges from 0 to 1 . Per DisGeNet, a
score of 0.1 corresponds to an average of about 3 sources of evidence. A score of 0.25
corresponds to between 4 and 5 sources of evidence for single gene-disease
assocation.
Optimized AmpliSeq On Demand Genes
We developed a “Circle of Optimization” process for designing,
measuring, refining and redesigning gene Assay designs. We developed
a custom assay design pipeline to incorporate all available information.
Gene designs were manufactured , their performance was measured as
part of development pools, and then 10 representative disease panels of
sizes ranging from 17 to 300 genes (322 to 8320 amplicons) were created
and their performance was measured.
All genes are created as 2-pool designs, with a maximum amplicon length
of 275 bases.
Seamless Design of Ion AmpliSeq On-Demand Gene Panels based on Diseases or Gene List
Selecting genes based on browsing Disease hierarchy
We developed an interactive web site running on AWS to facilitate seamless, fast, comprehensive design of gene panels. This is accessible at
AmpliSeq.com. A custom AmpliSeq On-Demand gene panel can be built using the optimized assays from any subset or all of the selected genes.
Optimized gene panels can be developed narrowly targeted to specific diseases, or larger gene panels can be developed for broader phenotypes.
Browsing through the disease hierarchy reveals the number of genes associated with each disease at every level of the hierarchy. Any number of
diseases at any combination of levels can be selected, leading to a list of genes associated with each of the diseases, ordered by score. Genes
available pre-optimized on-demand can be selected or unselected to create an Ion AmpliSeq On-Demand Panel. Additional genes can be added
manually.
Designing panels based on Gene List
Alternatively, genes can be selected without going through the disease hierarchy by searching for genes of interest, or by uploading a gene list.
Panel Sizes offered
Panels can be ordered in two size ranges: 1-50 genes or 51-300 genes. The minimum number of amplicons is 24, maximum is 9000 amplicons.
Adding Spike-In Panels
Genes of interest that are not yet available on-demand can be ordered in a separate panel that can be spiked in to the Ion AmpliSeq On-Demand
panel. The spike-in panel will be custom manufactured and is available in large reaction sizes.
Observed Performance by base, amplicon, gene, with IGV
All of the genes on-demand have empirical performance data from assay development and verification. This performance information is available on
AmpliSeq.com. IGV enables visualization of the genomic positions of exons, the amplicons that cover them, and the empirical observations of
coverage of each base and each amplicon for all genes in every custom Ion AmpliSeq On-Demand Panel. This enables confidence that amplicons
are designed to cover each important base and that observed performance is good for each base.
Analysis: Variant Calling through Annotation, Functional Impact, to Prioritization
Ion Reporter software provides a seamless end to end analysis for AmpliSeq On-Demand panels, including mapping, variant calling, and
CNV detection. Single sample, Trio, and Tumor-Normal pipelines are available. Pipelines include mapping and variant calling, followed by
functional annotation, protein prediction, and detailed annotation of variants and genes based on their presence in public databases with link-
outs to external sites. The software enables the essential steps of annotation, filtering, and prioritization, and finally reporting.
References
DisGeNET and Gene Scoring
http://www.disgenet.org/web/DisGeNET/menu/dbinfo#score
Level 1 Diseases Examples of Genes Associated with each Disease
# of total
Genes
ACMG APC, MYH11, ACTA2, MYLK, TMEM43, DSP, PKP2, DSG2, DSC2, BRCA1, BRCA2, SCN5A,, WT1 … 59
Newborn Screening
HBB, CYP21A2, MMACHC, MMADHC, KCNQ1, HPD, GJB2, MTHFR, TAZ, PCCB, PCCA, GCH1, QDPR, MCCC1, MAT1A, CBS, PTPN11, TAT, GJB3,
MTRR, TPO, TSHR, DUOX2, FAH, GNMT, GJB6, AHCY, MTR, TG, G6PD, IDUA, HBA1, SLC25A13, PAX8, HBA2, SLC5A5, CPT2, PTS, PCBD1, DECR1,
MCCC2, TSHB, ACADSB, HADH, MMAA, OPA3, ACADVL, AUH, DNAJC19, HMGCL, HSD17B10, MCEE, MMAB, MUT, CFTR, JAG1, FBN1, ZFPM2, ARSB,
GAA, ABCD1, IDS, KCNH2, NKX2-5, KCNJ2, LAMP2, ACADM, GUSB, BTD, ASS1, DNAI1, SLC22A5, ACADS,,.
276
Cardiovascular Diseases NPPA, ZFPM2, TGFB1, TGFBR1, TTN, STAT3, FLT1, TMEM43, HLA-A, MYLK, IGF2, AVPR1A, NPHS1, MYL3, HOXA1, PDE8B… 1375
Congenital, Hereditary, and
Neonatal Diseases and
Abnormalities
MECP2, TP63, VHL, LHCGR, XPC, RASA1, GNAS, KCNH2, MFN2, ACADVL, HBA1, DKC1, IVD, PITX2, CYP1B1, RHO, SMAD3, KRAS, RDH12, TFAP2A,
HMOX1, ABCB1, GCLC, CBFB, DNAH5, SLC40A1, APP, ETFDH, PSEN1, SYNE2, CCDC40, GNA11, MYL3, BARD1... 1743
Digestive System Disorders APC, MTHFR, SLC37A4, ABCB4, FH, F2, ARID1A, NOTCH2, C3, PRKDC, KCNQ1, IL7R, PEX5, POLG, PALB2, SLIT2, GLI3, FBN2, FOXA1, IRX5,
CARD11... 1660
Disorder of eye KIT, OPTN, SOD2, NOS3, FKRP, TGFBR2, GATA6… 473
Endocrine System Diseases TP53, CYP11B1, COL3A1, NR0B1, RECQL4, CHEK2, PIK3CA, EP300, BMP2, BAP1, SERPINA1, RYR2, BARD1, DPYD, SHOC2, APOE, ACTG1,
MAML3… 982
Female Urogenital Diseases
and Pregnancy Complications
FSHR, SCNN1B, HSD17B3, PPARG, HLA-B, MMP2, NR3C1, DICER1, SLC46A1, CSF1R, IFT122, SERPINA1, POLG, FANCA, VCAN, INF2, FAT4, KDM6A,
CSMD3… 2085
Hemic and Lymphatic
Diseases
FLT3, TBX1, TP53, TGFB1, ATM, MTR, PPARG, CFI, IGF2, AKT1, CTNNB1, RET, RP1… 659
Immune System Diseases VWF, WT1, PTPN11, TERT, FLT4, CHEK2, LHCGR, CASP8, HSD3B2, MAPK1, MID1, TLX1, EPHX2, TFR2, TRAF1, CALR, NPPA, FGFR4, GH1, DMD,
NKX2-5, DNAH5, IGSF1… 1620
Male Urogenital Diseases ESR1, CHEK2, APC, POLG, HSD17B3, NFE2L2… 1741
Mental disorders PAX6, NF1, COL11A2, PAX3, GNAQ, ZEB2, KCNH2, CYP11B1, PTGS2, FRAS1, CYP11B2, KCNE2, GH1, DLD, HFE, APC, EYS, PSEN2, MTRR, ESR1,
HAND2, FADD, CFI, FAS, APOB, ETV6, STAT1, PRDM1, TLX1, DEFA5, NKX2-6… 1355
Musculoskeletal Diseases
COL1A1, NOS3, POMC, RET, MMP2, GHR, VDR, TFAP2B, NKX2-5, CACNA1A, RAF1, CHEK2, NRXN1, FLG, PTGIS, HP, MLC1, EDARADD, KRAS, HLA-
A, NTF3, CYP2C9, LTBP2, SYNE1, BRIP1, CASP1, BAP1, BARD1, PROKR2, BMP6, HBA1, PEX13, IRS1, MYO6, RUNX1, MAMLD1, SPG7, SLC40A1,
MYL3, KRT6B, RBM20...
1117
Neoplasms PTEN, KRAS, PTCH1, CREBBP, XPC, CHEK2, WFS1, HBA1, GATA2, TSHR, ARID1A, SMO, CDKN2B, CP, CSF1R, PIK3CG, MUC5B, FOLR1, IFT122,
SETD2, ACTN4, CALM2, SLIT2, SF3B1, DSG2, SLC29A3, TPMT, FSHR, SGCB, MAGEC1, PARP4, RAD21, MYOT, TPR, BCLAF1, HSD17B10… 2851
Nervous System Disorder KCNQ1, RET, SLC26A2, EYA1, SOD1, TP53, SOD2, COL3A1, CYP1B1, BLM, EDA, HSD17B3, KCNE1, MAP2K2, SOX2, GATA6, LRRK2, DBT, IRX5,
AVPR1A, CYP11A1, SETBP1, CASP1, PMS2, SLIT2, CACNA1S, MAGEC1, CIC, CBFB, ABCG2, TG, CDH1, KRT6A, MUTYH, TAT, BTD, CHD4… 2186
Nutritional and Metabolic
Diseases
FGFR3, NOS3, TGFB1, SLC46A1, OCA2, ACADVL, SERPINA1, RAG2, DLD, GALE, IKBKG, MUT, EPHX2, FTL, PON2, NOD2, AVPR1A, ITIH4, MYLK,
KCNJ18… 1317
Otorhinolaryngologic Diseases DSPP, PIK3CA, TNF, COL2A1, TJP2, KRT6B… 195
Respiratory Tract Diseases NOS3, ZFPM2, ESR1, TGFBR2, LPIN2, NFE2L2, TNC, APOC3, ANK2, ATF3, IGF2R, MYLK, MECP2, ACTN2, COL1A2, PCSK9, BSN, PTCH1, FOXC2,
ACTG1, DYX1C1, SYNE2… 804
Skin and Connective Tissue
Diseases
MECP2, WT1, COL5A1, MPZ, SOX9, NPPA, FGD1, CHD7, LYST, TLR4, HNF1B, RHO, KCNE2, MAP2K1, FH, TAZ, PRKCSH, RDH12, MITF, DPYD,
IFT140, LRRK2, ATF3, CBFB, IFT88, DES, SYNE2, SRGAP3, SMARCB1, SMAD2, ESPN, BCAT2, CSF1R, MAP3K1, FGFR4, HABP2, SGCB, STXBP1,
SRPX2, DRC1...
1547
Stomatognathic Diseases HLA-B, PCSK5, FAT1, KRT6B… 298
Disease Categories and Associated Genes and Gene Panels
Examples of Genes associated with the Disease Research areas at Level 1 of the hierarchy. There are >3000 AmpliSeq On-Demand genes as
of November 2017. Optimized gene panels can be developed narrowly targeted to specific diseases, or larger gene panels can be developed
for broader phenotypes. Disease categories include early onset neonatal phenotypes such as metabolic disorders, Severe Combined
Immunodeficiency (SCID), heme disorders; and late onset disorders such as cancer predisposition and cardiovascular disorders.
For Research Use Only. Not for use in diagnostic procedures. © 2017 Thermo Fisher Scientific Inc. All rights reserved.
All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
Panel Based Uniformity: uniformities of libraries prepared from matched
frozen (darker bars) and dried blood samples (lighter bars) for two
example panels; a hematology panel with 136 genes, and a Rheumatoid
Arthritis panel with 30 genes. Sample input was 10 ng. Error bars
represent ± 1 SD
Sequencing the Panel with the Ion S5 System
Ampliseq libraries are run on Ion sequencing instruments, including
the Ion S5 System. Sequence from DNA library to data is available in
as little as 24 hours with only 45 minutes of total hands-on time.
This integrated suite of analysis tools from disease area to gene assay design to variant analysis, together with optimized assays, enables robust
and fast development of custom panels. This full end-to-end solution streamlines the process selecting genes associated with disease research
areas of interest; creating a panel for next-generation sequencing with these genes with amplicons that have already passed through optimization
and performance verification; sequencing samples with this panel using as little as 10ng of input DNA; and performing automated, straightforward
data analysis of the DNA sequence through to SNP detection, annotation, filtering and prioritization.
Gene
Design
Complete