Technical Note
Enrichment Workflows for Single Cell Genome Analysis
Authors
Jon Zawistowski, PhD
Senior Director
Research & Development
BioSkryb Genomics, Inc.

Durga Arvapalli, PhD
Scientist I
Research & Development
BioSkryb Genomics, Inc.

Swetha Velivela, PhD
Scientist I
Research & Development
BioSkryb Genomics, Inc.

Isai Salas-Gonzalez, PhD
Computational Biologist
Bioinformatics
BioSkryb Genomics, Inc.

Jay A.A. West, PhD

President, CEO, Co-founder
BioSkryb Genomics

Integration of Targeted Enrichment Methods for Single-cell
Genomic Studies Using the ResolveDNA Product Solution
BioSkryb Genomics has established multiple workflows to enable low DNA input and single-cellgenomics. These workflows highlight how next generation amplification, library preparation andsequencing technologies yield high quality single-cell targeted panel data.

Introduction
Heterogeneity within and between
cellularpopulations
dictates
the fate of all tissues in bothnormal development and disease
pathogenesis. The development of
single-cell approaches has revealedamazing diversity across tissues.
While the majority of methods
to define cellular variation arebased on the transcriptomics1,2,
ascertaining
genomic
diversityenables the understanding of theunderlying blueprint of cellular
heterogeneity. Numerous biologicalstudies have demonstrated that
accurate identification of genetic
variation in single cells is essential forunderstanding the role of mutationin normal development and in
disease3,4,5. Key to detecting thisdiversity is the ability to faithfully
replicate the genomes that are notdetectable reliably at the single celllevel without prior amplification. The
development of Primary Templatedirected Amplification (Figure 1)
for individual cells and low DNA
inputs allows for amplification withunprecedented uniformity, providing
revolutionary sequencing breadthand sensitivity6.

Genomic DNA

Primer (6-9mer)

Amplicon DNA

Termination base

Isothermal Polymerase

Primer Annealing and Polymerase Complexing
PrimerPrimerPrimerPrimer Extension and Termination

250-1500 bases

250-1500 bases

Primary template
reprimed for additional
amplification (favored)

250-1500 bases

Further amplification inhibited
due to short size

250-1500 bases

Genomic DNA

Short fragment DNA pool created ~ 500K fold amplification

Figure 1. The principle of PTA. ResolveDNA provides unbiased amplification
by utilizing random priming combined with a novel approach to produce a true
representation of original sample template. The use of proprietary nucleotides
prevents the production of long amplicons, which are kinetically unfavored
to be re-copied during the amplification reaction. By limiting the size of theproduced amplicon, primers are re-directed to the primary template.

While whole genome sequencing (WGS) using next generation sequencing (NGS) uncovers striking
single nucleotide variation (SNV) across the entire genome, much of intergenic and intronic regions
remain unannotated. Due to this and the cost of WGS, more cost effective approaches, such as wholeexome sequencing, (WES) are desirable. WES has broad coverage of all annotated and expressed genes
and is able to interrogate the impact of genetic variation. Alternatively, user-selected specific regions ofthe genome for high depth NGS analysis provide a cost effective approach allowing increased cellular
For Research Use Only. Not for use in diagnostic procedures.

An Integrated Workflow for Single-cell Genome Analysis lysis

212

3456

Single-cellIsolation

ResolveDNA
Whole GenomeAmplification

ResolveDNA
BeadPurification

Enrichment(TruSight Panel)

Next
Generation
Sequencing

BaseJumper
Analysis

Enrichment(Exome)Enrichment(xGen Exome)

Figure 2. ResolveDNA™ Targeted Enrichment Workflow: Isolated single cells undergo Primary Template-directed Amplification, followed by tagmentation orenzymatic-fragmentation-based library preparation. Tagmentation-based libraries can then undergo hybrid capture with Illumina TruSight panels or with IDT
exome panels, while enzymatically fragmented libraries are compatible with TWIST and IDT exome probe sets, library preparation, next generation sequencing, andanalysis with BaseJumper software.

throughput and broad genomic analysis of a subset of genes
to the entire exome. As a result, BioSkryb Genomics has paired
several downstream enrichment products (Figure 2) with ourResolveDNA8 whole genome amplification product solution forboth experimental models and clinical samples9.
Each workflow provided here, is further detailed in a discrete
BioSkryb Genomics Technical note which will guide the user
on the process of generating enriched NGS-ready libraries for
single cell genomic studies. All Techincal Notes can be found at
BioSkryb.com.

 Illumina DNA Prep for Enrichment
A workflow demonstrating both WGS and genome
enrichment using Illumina DNA preparation for Enrichmentwith the TruSight One Expanded panel containing 6700
genes, with ~16.5 megabase of coverage in the genome(~0.5% of the comprehensive genome).

 Twist BioSciences Human Core Exome Enrichment
A workflow which utilizes double-stranded DNA (dsDNA)
probes within a comprehensive target enrichment kit forexome and targeted sequencing. Using dsDNA as opposed
to single-stranded DNA captures all specified sequences
uniformly. The TWIST Human Core Exome covers nearly theentire exome (~34 megabases; ~1.0 % of the comprehensivegenome).

 Integrated DNA Technologies V2 ExomeTo provide increased depth of coverage and enable highmultiplexing of samples, the xGen Exome Research Panel v2
targets only the coding sequences of human coding genes
in the RefSeq 109 database. The xGen Exome Research Panelv2 consists of 415,115 individually synthesized and qualitycontrolled xGen Lockdown Probes. The panel spans a 34
Mb target region (19,433 genes) of the human genome andcovers 39 Mb of probe space.
© 2021. BioSkryb, Inc. All rights reserved.

References
1. Pollen, A.A., et al., Low-coverage single-cell mRNA sequencing reveals cellular
heterogeneity and activated signaling pathways in developing cerebral cortex.
Nat Biotechnol, 2014. 32(10): p. 1053-8.
2. Nowakowski, T.J., et al., Spatiotemporal gene expression trajectories reveal
developmental hierarchies of the human cortex. Science, 2017. 358(6368): p. 13181323.
3. Navin, N.E., Delineating cancer evolution with single-cell sequencing. Sci Transl Med,2015. 7(296): p. 296fs29.
4. Xu, X., et al., Single-cell exome sequencing reveals single-nucleotide mutation
characteristics of a kidney tumor. Cell, 2012. 148(5): p. 886-95.
5. Alexander, J., et al., Utility of Single-Cell Genomics in Diagnostic Evaluation ofProstate Cancer. Cancer Res, 2018. 78(2): p. 348-358
6. de Bourcy, C.F., et al., A quantitative comparison of single-cell whole genome
amplification methods. PLoS One, 2014. 9(8): p. e105585.
7. Gonzalez-Pena, V., et al., Accurate genomic variant detection in single cells withprimary template-directed amplification. Proc Natl Acad Sci U S A, 2021. 118(24).
8. BioSkryb Genomics, i., ResolveDNA™ Whole Genome Amplification Kit For high-quality
single-cell and low-input DNA amplification, in www.bioskryb.com, B. Genomics,Editor. 2021, BioSkryb Genomics: Durham, NC. USA
9. Zawistowski, J., et al., Single-cell oncogenic mechanistic heterogeneity defined by PTA
in primary Ductal Carcinoma In Situ, in Application note, Bioskryb Genomics, Editor.
2021, BioSkryb Genomics: www.bioskryb.com. p. 1-5.

For more information or technical assistance:
info@bioskryb.com
Published by:

2810 Meridian Parkway, Suite 110
Durham, NC 27713
www.bioskryb.com
All data on file.
For Research Use Only. Not for use in diagnostic procedures.
BIOSKRYB and RESOLVEDNA are trademarks of BioSkryb, Inc.
All other product names and trademarks are the property of theirrespective owners.
TAS-028, 01/2022