APPLICATION NOTEROBUST VARIANT CALLING WITH SRSLY®
USING THE HORIZON ONCOSPAN cfDNA
REFERENCE STANDARD
INTRODUCTION
Cell-free DNA (cfDNA) in blood originates predominantly from apoptosisof varying blood cell lineages1,2. In patients with cancer, a small fractionof the cfDNA known as circulating tumor DNA (ctDNA) originates fromdying tumor cells3. Recently, cfDNA variant calling has emerged asa noninvasive next-generation sequencing (NGS) method to monitor
disease progression and treatment efficacy.
Claret Bioscience has developed an easy and efficient NGS preparation
method called SRSLY that features an optional unique molecular
Identifier (UMI) add-on step for accurate detection of rare ctDNAvariants4. UMIs are short molecular barcodes that are used to uniquely
tag each molecule in a sequencing library to aid in the detection of
single nucleotide polymorphisms (SNPs) and resolve polymerase
chain reaction (PCR) duplicates5,6. In analyses of NGS data from cancer
samples, this allows true pathogenic variant alleles to be distinguishedfrom variants introduced by copying errors anywhere in the library
preparation and sequencing process.
Here we show that SRSLY library preparation can be used to detectcommon cancer variants at low allele fractions from varying sample
input amounts. We use Horizon Discovery’s OncoSpan cfDNA standard,a well-characterized genomic reagent that can be used for detectionof variants with known variant allele frequency (VAFs) and Twist
Bioscience’s custom Pan Cancer targeted-enrichment Panel. Targetedenrichment allows for cost effective sequencing of the portions of thegenome of clinical interest, including low frequency variants.

123Highlights• SRSLY is a unique NGS library
prep kit from Claret Biosciencethat renders all input DNA singlestranded prior to adapter ligationand captures all DNA strands
even when nicked or damaged• Here we showcase SRSLYs UMI
add-on kit’s ability to call low
AF ctDNA variants using
Horizon’s OncoSpan cfDNA
standard and Twist’s PanCancer
enrichment panel• The Horizon OncoSpan cfDNA isa valuable tool for evaluation ofNGS library preparation methods• Compared to traditional dsDNAlibrary preps SRSLY exhibits
increased library complexity andsimilar UMI error rate and VAF
calling sensitivity at all input
amounts tested

45Input

Library Prep

Enrichment

Sequencing

Analysis

Oncospan cfDNA
5-20ngSRSLY® + UMI or
DS Prep + UMI

Pan Cancer Panel(800kb)Illumina®
2x150bp

UMI correction
Complexity
SNP Calling

1

APPLICATION NOTEMETHODS
Library Preparation and Sequencing
1. DNA input pool
Fragmented dsDNA
with overhangs
HO 5’

3’ OH

3’ssDNA

Damaged (Nicked) dsDNA

5’FragmenteddsDNA with blunt ends2. Denature and capture
as single-stranded DNA

Using the Horizon OncoSpan cfDNA standard, eight
libraries were prepared for NGS. Three libraries wereprepared using 5, 10, 20 ng of the cfDNA standard withClaret Bioscience’s SRSLY Library Prep kit with UMI
Addition (Figure 2). For the same input amounts, libraries
were also prepared with a traditional double-strandedlibrary prep kits with UMIs (DS prep). The libraries werethen pooled and enriched using a custom Pan-Cancer
panel from Twist Biosciences® which spans ~100kb. Of
the 25 variants found in the Oncospan standard, 21 arecovered by the Twist enrichment panel. Enriched libraries
were sequenced on an Illumina HiSeq X at a read lengthof 2 x 151 bp following manufacturer’s instructions to adepth of >20 million reads per library (average of x foldcoverage for the 800Kb panel).

ssEnhancer

5’

5’
3’

HO

3’ OH
3’ OH
5’

The Horizon OncoSpan cfDNA standard is a wellcharacterized cell line-derived reference standard. This
standard contains >375 variants from over 152 key cancer
genes present at between 1-100% variant allele fraction(VAF), 25 of which are verified by ddPCR. These verifiedvariants include 8 indels and 17 SNPs. These 25 variants
were analyzed in this application note.

3. Phosphorylate template
DNA and llgate adapters
Block Forward
Block

Reverse Block
Block Block

Block

4. Add UMI and indexing
adapter via extension
Adapter2
i7 index

Adapter 2'Sequencing data was converted to FASTQ format withbcl2fastq 2.20.0.422, reads were downsampled to 20
million reads per library, and adapters were trimmed withSeqPrep2. Reads were aligned to hg38 with bwa mem

Flow cell bind UMI

Informatic Processing

To Index PCR

Figure 2. SRSLY library preparation protocol with UMI Addition.
Libraries are generated in 4 simple steps (excluding Index PCR)

0.7.15 for paired end reads. UMI tools 1.0.1 and was used to correct and group UMIs prior to duplicate marking with GATK
PicardTools version 2.23.8. Correcting and grouping reads by UMI was also performed with fgbio 1.3.0 to determine UMI
error rate. Target panel analysis was performed using GATK Picard 2.23.8, and variants were called using lofreq version
2.1.5. Figure 3 shows the pipeline inputs and outputs.

bcl2fastq

bwa

UMITools

FASTQ + UMI

Raw UMIBAM

Corrected
UMI BAM

PicardDuplicatemarked BAM

lofreqFGBio

Base Error
Estimates

Variant calls

Figure 3 – Bioinformatic pipeline

2

APPLICATION NOTERESULTS
Library complexity
10.0

SRSLY
DS Prep

7.5

5.0

2.5

DS Prep 20ng

SRSLY 20ng

DS Prep 10ng

DS Prep 5ng

SRSLY 10ng

0.0
SRSLY 5ng

Estimated Library Size (Reads in Millions)

To demonstrate the ability of SRSLY to perform in
ctDNA variant analysis, we prepared UMI libraries from
Oncospan cfDNA with a range of inputs representativeof amount used in clinical analyses using both SRSLY
and DS prep, followed targeted enrichment. The libraries
were sequenced and processed by the bioinformatic
pipeline described in Figure 3. GATK Picard Tools
was used to estimate the number of unique molecules
present in the library based on the number of paired end
molecules observed and the number of unique pairs
observed. Estimated library size increased with input
amount for both prep methods, but SRSLY showed amarked increase at higher inputs, with approximately
double the estimated library size of the DS prep at 10 and
20 ng of input (Figure 4).

Library
Figure 4 – Estimated library size from GATK Picard tools. Libraries
at four different inputs were downsampled to 20 millions reads
for analyses. Picard estimates library size based on the numberof paired end molecules observed and number of unique pairs
observed. The estimated library size is the estimated number ofunique molecules present in the library.

Reads per UMI
UMI tools was used to group reads by UMI and mapping location to determine the number of reads sequenced foreach UMI (Figure 5). At lower inputs, both SRSLY and DS preps have a higher proportion of UMIs with multiple reads
per UMI, showing that higher cfDNA inputs are ideal for variant calling. However, the higher input libraries havemore UMIs with lower number of reads per UMI. A lower number of reads per UMI implies a higher diversity of reads
sequenced and therefore increased library complexity. For all inputs tested, SRSLY has a higher number of UMIs with
only one read, and a lower maximum number of reads per UMI, due to increased library complexity (Figure 5).
5 ng input

10 ng input

20 ng input

60000
75000
1e+05

50000

Prep

UMIs

UMIs

UMIs

40000

SRSLY
DS Prep

5e+04

20000

25000

000102030

Reads per UMI

40500e+00
0102030

Reads per UMI

4050

0102030

Reads per UMI

4050

Figure 5 – Number of reads per UMI and the count of UMIs with that number of reads across prep method and input amounts. Reads per UMI isdefined as the number of reads sequenced containing a given UMI, while count is the number of UMIs with that number of reads per UMI. A lower
number of reads for a given UMI is preferred.

3APPLICATION NOTE
Error Rate

0.0100Mean Error Rate

After UMI correction and grouping reads by UMI, Fgbio
was used to determine the consensus sequence foreach UMI that had three or more reads. The mean error
rate for each sample was then calculated from the error
rates of the reads with the same UMI (Figure 6). At lower
inputs, the mean error rates are roughly similar between
SRSLY and DS prep, however at the two higher inputs
SRSLY has a lower error rate.

SRSLY

DS Prep

0.00750.00500.00250.0000SRSLY 5ng

DS Prep 5ng SRSLY 10ng DS Prep 10ng SRSLY 20ng DS Prep 20ng

LibraryFigure 6 – Mean error rate from FGBio for each prep type andinput level. Mean error rate is determined by first calling consensus
sequences from reads with the same UMI, and then determining theerror rate of each read from that consensus sequence.

Variant calling
After sequencing, mapping, and UMI correction, variants were called using lofreq v 2.1.5 and output in variant callformat (VCF). VCFs from each sample were merged with the Horizon Gold Standard VCF containing the truth calls
to determine accuracy (Figure 7). SRSLY was able to accurately call both SNVs and indels at all input levels. At every
input, SRSLY had read data supporting all confirmed VAF variants in the Horizon standard, though not enough reads tosupport a call by lofreq. Of the 25 ddPCR verified variants in the Horizon standard, two variants with very low VAF are
not called by lofreq in any sample for either SRSLY or DS prep. For the two variants that were not called in any sample
by lofreq, VAF was calculated from the number of reads containing the variant aligned to the site and the total numberaligned to that site. This method was also able to detect additional variants in the Oncospan standard that were notconfirmed by ddPCR (not shown).
SRSLY 5ng

SRSLY 10ng

300600200Unique molecules

SRSLY 20ng
900400600

100200300000CallDS Prep 5ng

DS Prep 10ng

Correct
Incorrect

DS Prep 20ng

Variant type500
3007504003002000250100

000.00

0.25

0.50

0.75

1.00

SNP

500

200100Indel0.00

0.25

0.50

0.75

1.00

0.00

0.25

0.50

0.75

1.00

Expected Allele Fraction
Figure 7 - Depth of coverage and variant allele fraction in the reference standard color coded by whether the variant is called correctly. Point shapeindicates variant type. Depth of coverage is the total number of reads at the variant site regardless of whether they contain the variant. Variant allelefraction is provided by the horizon reference standard. The dashed grey line indicates, for each allele fraction, the number of unique molecules
required to have 80% power to get sequence 10 reads with the alternate allele.

4

APPLICATION NOTECONCLUSIONSRSLY’s UMI add-on protocol demonstrates excellent performance in variant detection applications. Compared totraditional library preparations, SRSLY libraries have slightly higher complexity and better polymerase fidelity during
PCR at higher inputs and provide similar variant calling performance at identical sequencing depths. These data also
demonstrate that the Oncospan cfDNA can be used as standard to evaluate and deploy SRSLY for cfDNA-based variant
calling in research use and clinical settings.

REFERENCES
1. Stroun, M., Lyautey, J., Lederrey, C., Olson-Sand, A. & Anker, P. About the possible origin and mechanism of circulating DNA apoptosis and active DNA
release. Clin Chim Acta 313, 139-142, doi:10.1016/s0009-8981(01)00665-9 (2001).
2 Stroun, M., Anker, P., Lyautey, J., Lederrey, C. & Maurice, P. A. Isolation and characterization of DNA from the plasma of cancer patients. Eur J Cancer Clin
Oncol 23, 707-712 (1987).
3 Schwarzenbach, H., Hoon, D. S. & Pantel, K. Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer 11, 426-437, doi:10.1038/nrc3066 (2011).
4 Troll, C. J. et al. A ligation-based single-stranded library preparation method to analyze cell-free DNA and synthetic oligos. BMC Genomics 20, 1023,
doi:10.1186/s12864-019-6355-0 (2019).
5 Meyer, M. & Kircher, M. Illumina sequencing library preparation for highly multiplexed target capture and sequencing. Cold Spring Harbor protocols 2010,
pdb prot5448, doi:10.1101/pdb.prot5448 (2010).
6 Kivioja, T. et al. Counting absolute numbers of molecules using unique molecular identifiers. Nature methods 9, 72-74, doi:10.1038/nmeth.1778 (2011).

5

APPLICATION NOTESRSLY®
Claret Bioscience’s SRSLY® NGS library preparation kits for Illumina® provide reagents and proprietary adaptersthat convert a variety of fragmented DNA inputs (as low as 10 nt in length) to sequence-ready molecules. The
kits are provided in a modular format that allow maximum flexibility in the choice of Index PCR primers and DNA
purification strategies.

CLARETBIO PRODUCT INFORMATION:
To order the SRSLY kits email at orders@claretbio.com
Kit

Reactions

Catalog Number

SRSLY PicoPlus (Input < 10 ng)

24 or 96 reactions

CBS-K250B-24, CBS-K250B-96

SRSLY NanoPlus (Input > 10 ng)

24 or 96 reactions

CBS-K150B-24, CBS-K150B-96

Unique Dual Index Primers

24 or 96 reactions

CBS-UD-24, CBS-UD-96

Clarefy Beads

24 or 96 reactions

CBS-BD-24 or CBS-BD-96

Horizon Discovery offers a catalogue of reference standards including FFPE, cfDNA and gDNA controls fordiverse applications in next generation sequencing.

HORIZON PRODUCT INFORMATION:
To order the Horizon kits email orders@horizondiscovery.com
Kit

AmountCatalog Number

Oncospan cfDNA

350 ng

HD833

P.O. Box 3052 | Santa Cruz, CA 95063 | +1.833.425.2738 | www.ClaretBio.com | info@ClaretBio.com
Copyright © 2022 Claret Bioscience. All rights reserved. 03042022

6