Sequencing

11th Annual Sequencing, Finishing, and Analysis in the Future Meeting
DEVELOPMENT OF AMPLICON PANELS FOR
TESTING OF INHERITED MENDELIAN DISEASES
Wednesday, 1st June 17:45 La Fonda Ballroom Tech Talk (TT‐1.04)
April Lewis, Radmila Hrdlickova, Jiri Nehyba, Carrie Firmani,
Dylan Fox, Colby Clear, Masoud Toloue
Bioo Scientific
Single gene disorders follow a pattern of Mendelian inheritance where a single mutation in one gene
will cause disease. Objectives for screening of Mendelian diseases range from confirmation of clinical
diagnosis and determination of appropriate therapies, to assessment of disease reoccurrence risk,
carrier testing for at‐risk family members, and prenatal diagnostics. Disease studies of this nature can
be aided through the use of next generation sequencing (NGS). Custom amplicon‐based assays for
targeted NGS offer researchers an efficient solution for variant discovery, identification and
characterization on genes related to particular diseases.
Bioo Scientific offers amplicon design and panel development as a custom service with the use of NEXTflex
Amplicon Studio, proprietary primer design software. Compared to several other soft‐ ware programs,
NEXTflex Amplicon Studio has well‐defined primer selection criteria, and variable measurements are more
accurate and precise. The software is more flexible in its ability to cater to different experimental
requirements, allowing for the ability to offer custom services. Only the most optimally designed primer
mates, as quantified through a novel scoring system, were introduced into these panels.
Bioo Scientific has also developed an innovative library preparation protocol for these assays, which
embodies several new technical elements increasing efficiency and decreasing biases.
The proprietary software and innovative library preparation protocol were used in the production of 21
diagnostic gene panels for testing of predisposition to recurrent fever syndromes, cystic fibrosis, diabetes,
obesity, breast and colon cancers, and a variety of other genetic disorders.
The panels have 100% target region coverage. Target design covers all coding sequences of canonical
isoforms, as well as coding regions of a majority of alternative isoforms of 58 genes. Additionally,
targets cover 5‐50 bp (at least 25 bp in 56 genes) of flanking intronic sequences, several deep intronic
regions, and promoter regions where known pathogenic mutations were reported. In total, gene
targets make up 190.3 kb.
Primers were specifically designed for use with DNA from fresh or frozen samples to limit the number
of amplicons, and therefore increase coverage uniformity and multiplexing capabilities of all panels.
Consequently, amplicon inserts range from 60‐251 bp. Total amplicon size was kept under 280 bp to
allow for compatibility with the Illumina MiniSeq instrument. However, these panels work with all
other Illumina and Ion Torrent platform Bioo Scientific provides complete kits with all reagents
required for library preparation, including size selection beads and 384 Illumina‐compatible barcodes
if desired. The amplicon panels follow a rapid and user‐friendly workflow, were validated by
sequencing, and are optimized to deliver high coverage uniformity and target specificity.
Development of severe diseases and pathological conditions are often associated with inherited mutations
in a single gene. Therefore, screening for these mutations is vital in determining treatments,
assessing familial pedigrees for risk, and potentially aiding in preventative care. With distinct attention
to primer design, target definition, and simplicity of library preparation, Bioo Scientific’s
amplicon panels offer a cost‐effective and complete solution.
30