An Automated Workflow that Enables Cell-Free DNA Extraction
& Whole-Genome NGS Library Preparation from 24 Plasma Samples
in a Single Workday
M. Carter*, M. Goldrick, Davina Kruczek, Mareike Honickel &
Arvind Kothandaraman*[email protected]
Bioo Scientific Corporation7050 Burleson Rd.Austin, TX 78744
OVERVIEWStudies reported here demonstrate an integrated approach
for automated cf DNA extraction from 24 plasma samples in less than
one day or 96 samples in approximately two days. We also highlight
the results of our automation workflow when analyzing the size
distribution of fetal-derived cf DNA in maternal circulation. The
research tools and approaches we have developed for high-throughput
cf DNA extraction and analysis will facilitate the use of plasma
and other biofluids for an even wider range of non-invasive and
cost-effective clinical research applications.
METHODSPlasma Samples. Blood was collected by a commercial
vendor from healthy donors into bags containing K2-EDTA
anti-coagulant. Plasma was separated from RBCs and WBCs by
centrifugation at 1,600xg for 10 minutes, carefully removed, and
shipped to Bioo Scientific® on wet ice. Plasma was further spun at
4,000xg for 20 minutes. The double-spun plasma was removed and
pooled where necessary. Donors 1 and 4 are pools of various donors
while Donors 2 and 3 are individual donors. Plasma from K2-EDTA
whole blood from the second trimester pregnant donor was obtained
from a commercial source (Zen Bio®), and plasma from the
first-trimester pregnant donor was obtained from blood collected in
Streck BCT tubes and fractionated, using a double-spin protocol.
Both pregnant donors carried male fetuses.
Plasma DNA extraction and quantitation. cf DNA was extracted
from 24 X 5mL plasma replicates for each donor (or donor pool)
using the NextPrep-Mag™ cf DNA Automated Isolation Kit and script
(Bioo Scientific) on the chemagic™ 360 automation platform
(chemagen™). The cf DNA was eluted in 80 µL. Concentration of
eluted cf DNA was determined by Qubit® dsDNA HS assay (Thermo
Fisher Scientific®) using a 1:20 dilution. Concentration and size
distribution of the cf DNA samples were characterized using the
High Sensitivity DNA kit and 2100 Bioanalyzer® platform
(Agilent®).
Library preparation for whole genome sequencing. An equal volume
(32 µl) of each extracted cf DNA was transferred to a 96 well
hard-shelled PCR plate and used as input for whole genome library
preparation on the Sciclone® G3 NGS workstation. Libraries were
made using the NEXTflex® Cell-Free DNA-seq kit for Illumina®
platforms, with barcoded adapters (Bioo Scientific®) diluted 1:8.
Libraries were analyzed for yield and size distribution on the
LabChip® GX Touch™ HT platform (PerkinElmer®). Libraries from the
pregnant donors were sequenced on the Illumina®
MiSeq® platform as a 2x150 sequencing run and analyzed. Note
that cf DNA library preparation does not include a DNA
fragmentation step since the DNA is naturally fragmented to a size
of ~170 bp.Bioinformatics. Paired sequencing reads were trimmed off
3’ adapter sequences using Cutadapt version 1.9.1 and aligned to
human genome GRCh38 using Bowtie2 version 2.2.6. Y chromosome
mapped reads were copied to additional alignment files by using
Samtools version 1.3.1 view command. Genome and Y chromosome
alignment files were converted back to sequencing files using
Picard-Tools 1.95 in preparation for insert size binning. Binning
of insert size on aligned sequencing reads were done using custom
Python2 scripting, utilizing Biopython 1.66 (Python™). Binned
insert sizes were stored in comma-separated values (or .csv) files
and were graphically analyzed using Excel 2016. The % of reads
mapping to the human Y chromosome were used to determine the fetal
fraction of cf DNA.
RESULTS
Figure 1. Automation workflows for 24 and 96 samples from 5mL
plasma to sequencing-ready libraries. (A) Material s used from left
to right: (1) chemagic™ 360 instrument for automated cfDNA
extraction from 5mL plasma; (2) NextPrep-Mag™ cfDNA extraction
reagents; (3) Sciclone G3 NGS workstation for automated library
prep; (4) NEXTFLEX® Cell-Free DNA-Seq lbrary prep reagents. (B)
Single day workflow for 24 samples. (C) Two day automated workflow
for 96 samples
INTRODUCTIONcf DNA is a fluid sample type with vast applications
in the analysis of cancer-associated sequence variants for the
detection of actionable mutations in patients being treated for
malignant disease, which can enable earlier treatment at a lower
cost, monitoring cancer-linked cf DNA variants in healthy
individuals for early detection of malignant disease, non-invasive
prenatal diagnostics for early detection of genetic abnormalities
without the risk of fetal injury, monitoring of organ transplant
recipients for early signs of organ rejection, and tracking
pathologies that do not involve differences in primary genetic
sequence. To realize the full potential of cf DNA-based
diagnostics, clinical research is needed to improve workflows that
efficiently extract circulating cf DNA at high yield and purity,
convert the cf DNA into libraries to allow massively parallel
sequencing of regions of interest, and carry out the bioinformatic
analysis steps. This poster describes methodology developed at Bioo
Scientific, a PerkinElmer® Company, for both rapid high-throughput
automated extraction of cf DNA from high volumes of plasma (5 mL)
and for preparation of whole-genome cf DNA libraries from the
extracted cf DNA, in a fully automated fashion.
Figure 3. Automated Library prep of extracted cfDNA– 32µl of
extracted cfDNA shown in Figure 2 was used as a template for
library preparation using the NEXTflex® Cell Free DNA-Seq kit on
the PerkinElmer® Sciclone® G3 NGS Workstation. 96 samples were
processed in a single run. (A) LabChip® GXII Touch™ gel image of 96
libraries. DNA libraries were diluted 1:8 and run on a HSDNA chip.
(B) Nanomolar concentrations of the ~300bp region of the library
(corresponding to mononucleosomal cfDNA) was quantified using the
LabChip® GX Reviewer software. Box and whisker plots were generated
for each donor. %CVs were 11, 9, 28, and 14% for donors 1-4,
respectively.
Figure 4. (A) Blood was collected from 2 healthy pregnant donors
with known male fetus (20 week - EDTA; 12 week - Streck BCT®) and
doubly spun to remove contaminating cells. Automated cfDNA
extraction of 5mL of the resultant cell free plasmas was carried
out using Bioo Scientific’s NextPrep-Mag™ cfDNA Automated Isolation
reagents on the chemagic ™ 360 instrument (red traces) . Library
Prep was automated on the PerkinElmer® Sciclone® G3 NGS workstation
using Bioo Scientific’s NEXTflex® Cell Free DNA-Seq reagents, 10
cycles PCR, with and without using an upfront SPRI cleanup to
remove non-mononucleosomal DNA (>200bp) (blue, green traces).
Yields of library size ranges were quantified using the
Bioanalyzer® software and showed that ~95% of non-mononucleosomal
peaks were excluded from libraries when size selection was used.
(B) Library sequencing reads were aligned to human genome GRCh38
and binned to insert size to examine the effect of size selection
on mononucleosome and non-mononucleosome quantity. Y chromosome
inserts represent reads derived from fetal DNA. Binned inserts from
total genome mapped reads and Y chromosome mapped reads show size
selection excludes non-mononucleosomal reads from sequencing
data.
CONCLUSIONSHigh concordance was seen between cf DNA yield and
size distribution for replicate 5 mL plasma samples processed for
automated cf DNA extraction using the NextPrep-Mag™ cf DNA
automated isolation kit on the chemagic™ 360 platform. The extent
of donor to donor variation was much higher, as expected, than the
extent of variation between replicate samples from the same donor
or donor pool (Fig 2). Excellent reproducibility was also observed
for yield and size distribution of the 96 whole-genome libraries
produced on the Sciclone® G3 NGS workstation (Fig 3). Regarding the
analysis of cf DNA obtained from the two pregnant donors, our
results clearly show that fetal reads (reads mapping to the
Y-chromosome) are present across the whole size distribution of cf
DNA (Fig 4b). This result is surprising, in light of reports that
fetal cf DNA fragments are typically shorter than non-fetal
fragments (Chan et al, PNAS, doi 1615800113.). Our studies also
demonstrate feasibility of automating the entire workflow for NIPT
research, and the ability to detect informative levels of fetal cf
DNA in first-trimester plasma samples, even without size-selection
of the cf DNA or the resulting whole-genome libraries. The rapid
automated extraction of cf DNA from high-volume 5 mL plasma
samples, coupled with the capability of converting the extracted cf
DNA into whole-genome libraries on a second automation platform,
provides an integrated workflow that will enable efficient liquid
biopsy-based investigations relevant for cancer and non-invasive
prenatal assessment. Future studies will focus on methodological
advances to maximize detection of rare variants in plasma and other
biofluids, using automated workflows.
For research use only. Not for use in diagnostic purposes.
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(A) 20 week EDTA 12 week Streck BCT cfDNA Library + Size
Selection Library - Size Selection
Figure 2. Highly reproducible cfDNA extractions on chemagic™ 360
instrument. cfDNA was extracted from 5mL of doubly-spun EDTA plasma
from four healthy donors in four automated runs (24 replicates of a
single donor/run). Reproducible yields are shown (1) qualitatively
by gel and electropherogram and (2) quantitatively for both total
DNA (Qubit® HSDNA assay) and for mononucleosome peak (Bioanalyzer®
100-275 bp region). For Donors 1-4, %CVs were 10, 10, 12, and 14%
on the Bioanalyzer® and 12, 7, 9, and 18% on the Qubit®,
respectively. Donor 4 showed significant contamination with gDNA,
resulting in much higher Qubit® yields for that donor. Each
automation run takes 75 minutes, with 15 minutes of hands on time.
On graph, Qubit = Qubit® HSDNA assay and BioA = High Sensitivity
DNA kit on the 2100 Bioanalyzer® platform
a PerkinElmer company
