1. health and fitness
2. disease

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NOVEL IMPROVEMENTS TO THE ILLUMINA TRUSEQ INDEXED LIBRARY CONSTRUCTION, AMPLIFICATION
AND QUANTIFICATION PROTOCOLS FOR OPTIMIZED MULTIPLEXED SEQUENCING
GAVIN J. RUSH1, ERIC VAN DER WALT1, JACOB KITZMAN2, LIESL NOACH1, ZAYED ALBERTYN3, COLIN HERCUS3, CHARLIE LEE2, JAY SHENDURE2, JOHN F. FOSKETT III1, PAUL J. MCEWAN1
Reversed
1 KAPA BIOSYSTEMS, 600 WEST CUMMINGS PARK, SUITE 2250, WOBURN, MA 01801 | 2 UNIVERSITY OF WASHINGTON, DEPARTMENT OF GENOME SCIENCES, FOEGE BUILDING S-250,
BOX 355065, 3720 15TH AVE NE , SEATTLE WA 98195 | 3 NOVOCRAFT, C-23A-5, TWO SQUARE, SECTION 19, 46300 PETALING JAYA, SELANGOR, MALAYSIA
A M P L I F I C AT I O N
S A M P L E P R E PA R AT I O N
INTRODUCTION
L I B R A R Y Q UA N T I F I C AT I O N
K A PA L I B R A R Y Q U A N T I F I C AT I O N K I T F O R O P T I M A L S A M P L E M U LT I P L E X I N G
R AT I O N A L E F O R R E A L-T I M E H I G H F I D E L I T Y A M P L I F I C AT I O N O F N E X T- G E N E R AT I O N D N A S E Q U E N C I N G L I B R A R I E S
The same guidelines and standards for the University logo
0
Manual Gel Purification
Sage Science Pippin Prep
Yield of adaptor-ligated library molecules before size
selection.
Yield of adaptor-ligated library molecules after size
selection.
Sheared genomic DNA was prepared in bulk by nebulization and
identical starting material was used for each library (1 μg of gDNA from
S. aureus, E. coli, or M. tuberculosis). Libraries were constructed using
reagents and procedures (see detailed methods on left-hand side) from
KAPA Biosystems (green; 9 libraries), or using the Illumina TruSeq DNA
Sample Prep Kit and Low Throughput Protocol (orange; 15 libraries).
Libraries were quantified by qPCR before size selection using the KAPA
Biosystems Library Quantification Kit according to the recommended
protocol. The estimated percentage of starting material that was
converted to useful, adaptor-ligated (PCR-amplifiable) library molecules
is provided.
Starting with 1 μg of either S. aureus, E. coli, or M. tuberculosis gDNA,
we constructed duplicate libraries using reagents and procedures from
the KAPA DNA Library Prep Kit (see detailed methods on left-hand side).
Duplicate libraries were pooled, and the three resulting libraries (S. aureus,
E. coli, or M. tuberculosis) were each split equally before size selection
either by manual gel purification (Qiagen QIAquick Gel Extraction Kit) or
by Pippin Prep automated gel purification (Sage Science). Libraries were
quantified by qPCR using the KAPA Library Quantification Kit according
to the recommended protocol. The estimated percentage of starting
material converted to useful, adaptor-ligated (PCR-amplifiable) library
molecules is provided.
Approximate
duration
No details supplied in protocol
Note: This can negatively impact library diversity because it
will result in the availability of less than 20 μL of library for
the subsequent preparative PCR reaction.
20
25
30
35
Time (min)
40
0
45 000
45 000
40 000
40 000
35 000
35 000
Fluorescence
Fluorescence
Fluorescence
15
30 000
00
20 000
15 000
5
10
15
20
30
35
40
30 000
25 000
20 000
15 000
10 000
10 000
5 000
5 000
Here we demonstrate the use of the system to:
0
0 1 2 3 4 5 6 7 8 9 10 1112 1314 1516 17181920
0 1 2 3 4 5 6 7 8 9 10 1112 1314 1516 17181920
Cycle
Cycle
0
B
25
0 1 2 3 4 5 6 7 8 9 10 1112 1314 1516 17181920 21 22 232425 2627 282930 3132333435
Cycle
Real-time high fidelity amplification of next-generation DNA sequencing
libraries.
Cycle 12 Cycle 13 Cycle 14 Cycle 15 Cycle 16
Target concentration
1. Generate Illumina TruSeq indexed libraries comprising equimolar concentrations of 3 bacterial genomes spanning a range of average GC contents (S. aureus, 33% GC; E. coli, 51% GC; and M. tuberculosis, 65% GC).
2. Pool multiple Illumina TruSeq indexed libraries in equimolar concentrations for equal representation in sequence data.
(A) Libraries are amplified using a SYBR® Green-based real-time high fidelity PCR master mix.
Four wells of the PCR plate contain fluorescein reference standards representing a range of
distinct DNA concentrations. Reactions terminated between standards 1 and 3 represent the
optimal library amplification range, depicted here from cycle 10-14. (B) Gel image of a typical
library stopped at different amplification cycles. Low and high molecular weight artifacts increase
progressively with additional cycles . (C) Shown are typical amplification plots for reactions
terminated at the lower (left panel) or upper (right panel) bounds of the targeted concentration
range (grey box). Library amplification reactions should ideally be terminated anywhere within the
indicated target concentration range.
Primer Mix (10X)
Size separation using either:
1. Manual gel extraction, purification (MinElute Gel
Extraction Kit, Qiagen). Recover 20 μL library DNA.
2. Automated DNA size selection and collection system
(Pippin Prep, Sage Science). Recover 30 μL library DNA.
PROGRAM REAL-TIME THERMOCYCLER
5
10
15
20
25
30
35
40
DISADVANTAGES ASSOCIATED WITH THE EXISTING
TRUSEQ WORKFLOW
45 000
40 000
Approximate
duration
30 000
25 000
20 000
15 000
10 000
5 min
0 1 2 3 4 5 6 7 8 9 10 1112 1314 1516 1
7181920
Cycle
1 x 50 μL qPCR reaction per library
25 μL
5 μL
20 μL
15 min
Fixed cycle number amplification High fidelity real-time PCR
· Wastage of library material required for cycle number optimization leads to loss of library diversity.
· Variability in the nature
and/or concentration of the input DNA
can result in under or over-amplification exacerbating amplification
bias and artifacts.
· Longer protocol involves more time and labor.
Example of multiplexed real-time high fidelity amplification.
20 libraries, spanning a ~64-fold concentration range (6 cycles), were simultaneously
amplified and terminated after 14 cycles. 14 of the 20 libraries fall within the targeted
amplification range. The remaining 6 libraries could either be used as is, noting that they
may be outside the optimal concentration range, or they could be re-amplified individually
or in high- or low-concentration groups.
ADVANTAGES OF HIGH
FIDELITY REAL-TIME PCR
Cycle number optimization
·Inconsistencies in reaction volume scale-
up can cause variable results.
5 000
0
SET UP AND INITIATE PREPARATIVE HIGH
FIDELITY qPCR
KAPA HiFi qPCR MM TruSeq PCR Primer Mix Size selected library · Gel electrophoresis steps are not amenable to automation.
· Post-amplification gel
electrophoresis QC is not
amenable to automation.
·
Built-in real-time quality
metrics (concentration
range) for each amplified
DNA library.
Std 1
·Real-time PCR is amenable
to automation.
0.001
M. tuberculosis
30 min
S. aureus
Pause the themocycler and remove a small scale PCR tube
after each predetermined cycle and place on ice before
initiating cycling.
TERMINATE HIGH FIDELITY qPCR
Terminate qPCR reaction when the linear amplification plots
of samples fall between fluorescent standards 1 - 3 or within
targeted concentration range. This is a 4 cycle termination
window and enables single plate amplification of libraries
with up to a 16-fold difference in initial concentration.
S. aureus (33% GC)
· Precise control over PCR
cycle number required for
optimal amplification.
60 min
Run an aliquot of each small scale sample on an agarose gel
or BioAnalyzer chip to determine the optimal cycle number
to be used for preparative gel electrophoresis.
30 min*
CLEAN UP qPCR REACTION
PROGRAM THERMOCYCLER FOR
PREPARATIVE PCR
5 min
98° for 30 s
10 cycles* of:
98° for 10 s
60° for 30 s
72° for 30 s
*or as determined by
optional small-scale
cycle optimization.
72° for 5 min
SET UP AND INITIATE PREPARITIVE PCR
1 x 50 μL qPCR reaction per library
TruSeq PCR MM 25 μL
TruSeq PCR Primer Mix 5 μL
Size Separated Library 20 μL
CLEAN UP PCR REACTION
60 min
Use AMPure XP Beads and elute in 32.5 μL Resuspension
buffer, according to the TruSeq protocol.
3h 10 min
QUANTIFY AMPLIFIED LIBRARY USING ILLUMINA
QUANTIFICATION PROTOCOL GUIDE
20
20
15
QUANTIFY AMPLIFIED LIBRARY USING KAPA
LIBRARY QUANTIFICATION KIT
15
10
10
5
5
0
0
0.3
fracGC
0.4
0.5
0.5
0.6
fracGC
0.7
0.8
60 min
windowed depth vs %GC, M. tuberculosis
l_7_tru12 amp:kapa_hifi_q_early
windowed depth vs %GC, S. aureus
l_7_tru12 amp:kapa_hifi_q_early
1h 50 min
Starting with 1 µg of either S. aureus, E. coli, or M. tuberculosis sheared
gDNA, we constructed duplicate libraries using reagents and procedures
from the KAPA DNA Library Prep Kit (see detailed methods on left-hand
side). Duplicate libraries were pooled, and the three resulting libraries
(S. aureus, E. coli, or M. tuberculosis) were each split equally before
size selection either by manual gel excision and purification (Qiagen
QIAquick Gel Extraction Kit) or by Pippin Prep automated gel purification
(Sage Science). We set the Pippin Prep instrument to collect library
molecules in the 370 bp – 450 bp range including adaptors, and we
attempted to select the same range via manual agarose gel purification.
TruSeq adaptors are 121 bp in total, suggesting targeted insert sizes
were ~250 bp – 330 bp (broken lines). Paired reads (2 x 75 bp) were
used to determine actual insert sizes.
25
25
0.2
Use AMPure XP Beads and elute in 32.5 μL Resuspension
buffer, according to the TruSeq protocol.
30
*Note: the 5 second step at 72° enables termination of the
qPCR reaction (once the desired fluorescent intensity has been
achieved) before the next cycle of denaturation is initiated.
30
30
25
25
20
20
15
15
10
5
5
0
· Seamless integration
with qPCR-based library
quantification.
· Ideally, libraries should
be minimally amplified
to a low final concentration
(~3.5 μM), not detectable
· KAPA HiFi DNA
via standard gel Polymerase is less prone
electrophoresis.
to amplification bias due to high- or low-GC content.
0.3
fracGC
0.4
0.5
15
10
5
0
3.0%
windowed depth vs %GC, S. aureus
l_8_tru2 amp:ilmn
30
25
25
20
20
15
15
10
10
5
5
0
0
0.2
0.3
fracGC
0.4
0.8
windowed depth vs %GC, M. tuberculosis
l_8_tru2 amp:ilmn
30
0.5
2.5%
0.6
fracGC
0.7
12
14
16 18
20
22 24
26 28 30
32 34
-4 -2 0
2
Log (Concentration)
KAPA Library Quantification Kits comprise DNA Standards (six 10-fold dilutions) and 10X Primer Premix, paired with KAPA SYBR® FAST qPCR Kits to accurately quantify the
number of amplifiable molecules in an Illumina GA library. The 452 bp KAPA Illumina GA DNA Standard consists of a linear DNA fragment flanked by qPCR primer binding sites.
Quantification is achieved by inference from a standard curve generated using the six DNA Standards.
2.0%
1.5%
1.0%
0.5%
KAPA HiFi
KAPA
KAPA HiFi
TruSeq
KAPA HiFi
TruSeq
KAPA HiFi
TruSeq
Phusion
TruSeq
TruSeq
Truseq
6.0
9.9
13.6
13.6
14.2
13.2
0.8
26%
24%
22%
20%
18%
16%
14%
12%
Amplification method
Library preparation
Calculated doublings
Effect of GC content on coverage depth for libraries amplified using
KAPA HiFi qPCR Master Mix or Illumina TruSeq PCR Master Mix.
Indexed libraries were prepared from identical sheared S. aureus (33% GC; left panels) and
M. tuberculosis (65% GC; right panels) gDNA using either the KAPA DNA Library Prep Kit,
or the Illumina TruSeq DNA Sample Prep Kit, and then amplified using the indicated PCR
reagents before paired-end sequencing (2 x 75 bp). After filtering and aligning read pairs
to reference sequences, 250 000 read pairs were randomly sampled for each genome,
and scatter plots of mean sequence coverage depth vs. GC content were generated by
analyzing 250 bp windows.
TM
For the AT-rich S. aureus genome (left panel), none of the samples showed gross amplification
bias compared to the unamplified control sample. GC-rich M. tuberculosis sequences
(right panel) in the library constructed and amplified using Illumina TruSeq reagents are
under-represented in the sequencing data. In contrast, the library prepared using KAPA
Biosystems reagents yielded coverage across the range of GC-content that is almost
indistinguishable from that of the unamplified control, indicating that amplification with
KAPA HiFi qPCR Master Mix introduced minimal additional GC-dependent coverage bias.
Sample
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
End-Point PCR
10%
0.5
10
KAPA Library Quantification Kit Workflow.
28%
fracGC
8
Cycle
qPCR
0.7
6
3.5%
Library preparation
0.6
4
4.0%
Amplification method
0.5
y = -3.4496x + 36.24
R² = 0.9998
20
0.0%
0
0.2
Std 5 Std 6
25
4.5%
Calculated doublings
10
Std 4
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
-0.1
5.0%
windowed depth vs %GC, M. tuberculosis
l_8_tru4 amp:noAmp
30
100 150 200 250 300 350 400
VALIDATE IDEAL CYCLE NUMBER BY
PERFORMING GEL ELECTROPHORISIS
M. tuberculosis (65% GC)
windowed depth vs %GC, S. aureus
l_8_tru4 amp:noAmp
Std 2 Std 3
Library Sample
2
Fraction of genome not represented
E. coli
Accurate qPCR-based library quantification ultimately
depends on three factors: (i) the accuracy and
reproducibility of the standards used, (ii) the ability
of the DNA polymerase used in the qPCR to amplify
all adaptor-flanked molecules with equal efficiency,
and (iii) accurate and reproducible liquid handling.
KAPA Library Quantification Kits are rigorously tested
to ensure minimal lot-to-lot variation. In addition,
KAPA SYBR® FAST qPCR Kits are designed for high
performance, high-throughput, real-time PCR. The
kit contains a novel DNA polymerase engineered via
molecular evolution, resulting in an unique enzyme
optimized for qPCR using SYBR® Green I dye
chemistry. KAPA SYBR® FAST qPCR Kits are ideally
suited to library quantification applications, as they
support high-efficiency amplification of both AT- and
GC-rich targets, and of fragments up to 1 kb in length.
Time (min)
Load 50 μL of each fluorescent standard 1 - 4 (in triplicate)
per qPCR plate.
REMOVE SMALL-SCALE PCR REACTIONS AT
DESIRED PCR CYCLES
15 min
DNA Size Selection System
10
0
depth
Genome
0.002
Size distribution of library molecules after size selection
by manual agarose gel purification or automated gel
purification with Sage Science Pippin Prep.
· Reduced potential for cross-
contamination amongst samples.
5
Fraction of genome not represented
· Potential for cross-
contamination amongst samples on a single gel.
0
KAPA SYBR® FAST
qPCR Master Mix (2X)
Insert Size
· Reduction of low molecular weight DNA contamination reduces library artifacts.
Time (min)
75
Pippin Prep
50
Significant time and labor savings from quicker run times and automated elution.
70
5 000
depth
· Contamination of adaptor-
dimers and other low molecular weight artifacts leads to lower
data quality.
·
Manual gel
0.000
· Selects tight, accurate, and reproducible DNA fragment size ranges.
65
10 000
SIZE SELECTION
98° for 45 s
30 cycles of:
98° for 15 s
60° for 30 s
72° for 30 s
Data Aquisition
72° for 5 s*
98° for 30 s
16 cycles of:
98° for 10 s
60° for 30 s
72° for 30 s
5 min
15 min
0.003
60
Fluorescent Standard 1
depth
· Manual gel electrophoresis requires longer run time and more manual steps.
Automated elution of sample material into buffer compatible with downstream workflows (PCR amplification, flow cell amplification).
15 000
depth
· Actual DNA fragment length distribution is broader and less reproducible.
·
PROGRAM THERMOCYCLER
SET UP AND INITIATE SMALL-SCALE PCR
Proportion of read pairs
· Requires manual gel excision and downstream extraction
of the sample from the agarose gel.
AUTOMATED SIZE SELECTION
55
35 000
Method
MANUAL GEL EXTRACTION
50
20 000
0
OPTIONAL
The KAPA Biosystems library construction reagents and protocol
produced ~25-fold more adaptor-ligated library fragments from the
same amount of starting material than did the standard Illumina TruSeq
DNA Sample Prep Kit. Important differences between the two library
construction procedures assessed here include: Combined enzyme
and reaction buffer “master mix” formulations (TruSeq) vs. separate
enzyme and reaction buffer (KAPA); and the absence of an Ampure XP
Bead clean-up between A-Tailing and Adaptor Ligation in the TruSeq
Protocol.
Size selection by manual gel purification yielded ~4-fold more DNA than
size selection by automated gel purification using the Sage Science
Pippin Prep apparatus. To a large extent this may be accounted for by
the fact that the Pippin Prep method yielded a much narrower size-range
of library fragments (see below), despite careful manual gel purification
targeting an equivalent size range of library molecules.
45
Multiple Sample Workflow using KAPA
High Fidelity qPCR Kit for TruSeq Library
Amplification
SIZE SELECTION
Size separation:
1. Manual gel extraction, purification (MinElute Gel
Extraction Kit, Qiagen). Recover 20 μL library DNA.
40
Fluorescent Standard 2
Here we present a high fidelity, real-time PCR method for rapid and convenient enrichment
and amplification of libraries. The benefits of this approach include: 1) automatable workflows,
2) built-in quality metrics for each enriched library, eliminating expensive and time-consuming
post-enrichment gel electrophoresis, 3) precise control over the number of PCR cycles required
for optimal amplification, 4) a quality control metric for identifying inconsistencies in library
preparation, and 5) seamless integration with KAPA qPCR Library Quantification kits.
TruSeq Low-Throughput Library
Amplification Workflow
35
Norm. Fluoro
500
Illumina TruSeq Library Prep
25 000
depth
KAPA Library Prep
371 pM
(~0.49%)
30
30 000
depth
0
1 000
25
Fluorescent Standard 3
35 000
Fluorescence
5 000
20
40 000
No
Amplification
885 pM
(~0.46%)
15
Fluorescent Standard 4
KAPA HiFi q PCR
(“early”)
10 000
10
Cycle 11
1 500
C
Time (min)
5
45 000
TruSeq PCR
15 000
0
TruSeq Library
Prep
20 000
1 546 pM
(~2.09%)
A
KAPA Library
Prep
25 000
21 767 pM
(~11.3%)
2 000
High fidelity PCR is used to selectively enrich library fragments carrying appropriate adaptor
sequences and to amplify the amount of DNA prior to sequencing. During PCR enrichment of
libraries, a subset of library molecules are amplified with reduced efficiencies, introducing bias
and resulting in uneven sequence coverage. GC content is known to be an important factor
in NGS library amplification bias, and different PCR enzymes and buffer formulations are likely
to show individual strengths and weaknesses in this regard. Furthermore, such biases – along
with other artifacts such as PCR-induced errors, adaptor dimers, PCR duplicates, and chimeras
– are exacerbated by over-amplification, while under-amplification results in insufficient yields.
Inherent uncertainty in the outcome of end-point PCR often demands downstream validation
of library quality by electrophoresis.
TruSeq Library
Prep
30 000
Concentration by qPCR (pM)
Concentration by qPCR (pM)
Dramatic improvements in commercial Next Generation Sequencing (NGS) platforms have resulted in spectacular reductions in the cost-per-base of DNA sequencing. Until recently, the primary
focus for innovation has been on improvements to the core sequencing technologies, with optimization of sample preparation playing a secondary role. The exponential gains in sequencing capacity
have simultaneously led to higher sample throughput, placing increasing emphasis on the importance of improved library construction protocols for multiplexed sample sequencing. While major
commercial NGS systems all require the construction of similar libraries via analogous workflows, some protocols and/or reagents offer significant advantages over others, and end-users must choose
among numerous alternative methods and reagents for sample preparation. We re-sequenced the Staphylococcus aureus, Escherichia coli, and Mycobacterium tuberculosis genomes to compare the
standard Illumina TruSeq sample preparation reagents and workflow with a number of innovative improvements including: alternative library preparation reagents and protocols; automated fragment
size selection; real-time library amplification; amplification-free sequencing; and accurate qPCR library quantification for sample pooling and multiplexed sequencing.
Accurate quantification of the number of amplifiable molecules in a library is critical to the
ofSeal
sequencing
results
on the Illumina
applyoutcome
to the University
— including color
use, staging
and background
colors. in
Thelower
minimum
size requirement
is .5”.
Genome Analyzer next-generation sequencing platform. Overestimation of library concentration
results
cluster
density
after bridge
The University logo guidelines can be found on pages 13-15.
PCR. Underestimation of library concentration results in too many clusters on the flow cell, which can lead to poor cluster resolution. Both
January 2010, Version 2.2
33
scenarios result in suboptimal sequencing capacity. qPCR is widely regarded as the gold standard for accurate quantification of DNA libraries
as it is the only technique capable of measuring the number of amplifiable molecules. The broad dynamic range of qPCR also enables
accurate quantification of extremely dilute libraries.
KAPA HiFi
KAPA
KAPA HiFi
TruSeq
KAPA HiFi
TruSeq
KAPA HiFi
TruSeq
Phusion
TruSeq
TruSeq
Truseq
6.0
9.9
13.6
13.6
14.2
13.2
qPCR
End-Point PCR
Percentage of the M. tuberculosis genome not represented at all (top), or
at ≥5x coverage (bottom) when using various library construction and PCR
reagents.
Indexed libraries were prepared from identical sheared M. tuberculosis (65% GC) gDNA using
either the KAPA DNA Library Prep Kit, or the Illumina TruSeq DNA Sample Prep Kit, and then
amplified using the indicated PCR reagents before paired-end sequencing (2 x 75 bp). Libraries
were quantified before and after amplification using the KAPA Library Quantification Kit to
determine the number of doublings in each case. After filtering and aligning read pairs to reference
sequences, 250 000 read pairs (~8.5x coverage) were randomly sampled for each genome.
80%
2
76.5 nM
1.04 μL
2.35 nM
% of total assigned reads in lane
4
4
5.31 nM
15.00 μL
2.35 nM
5
5
51.09 nM
1.56 μL
2.35 nM
7
6
36.88 nM
2.16 μL
2.35 nM
9
7
36.69 nM
2.17 μL
2.35 nM
11
12
6.67 nM
11.94 μL
2.35 nM
33.87 μL
11.76 nM
60%
Totals:
40%
POOLED LIBRARY 2
0%
5
12
Conc. In Final Pool
1
20%
4
Vol. in Pool
Relative qPCR concentration
M. Tuberculosis
S. aureus
qPCR Concentration
POOLED LIBRARY 1
100%
E. coli
I lumina TruSeq Index
1
5
7
9
11
2
10
6
8
2
12
2
2
38.41 nM
2.67 μL
4.35 nM
6
5
51.98 nM
1.97 μL
4.35 nM
8
6
51.24 nM
2.00 μL
4.35 nM
10
7
52.05 nM
1.97 μL
4.35 nM
12
12
6.84 nM
15.00 μL
4.35 nM
23.62 μL
21.73 nM
Totals:
TruSeq Index
qPCR quantification enables equal
representation of different sample types within
indexed libraries.
For each index (TruSeq 4, 5, and 12) we constructed three
separate libraries (S. aureus, 33% GC; E. coli, 51% GC; and
M. tuberculosis, 65% GC). Individual libraries were quantified
using the KAPA Library Quantification Kit and for each index
the libraries were pooled to achieve equimolar representation
for each genome. The results indicate that quantification is
reliable for samples with a wide range of GC content.
qPCR quantification enables equal representation of pooled indexed libraries.
Eleven indexed Illumina TruSeq libraries were quantified by qPCR using the KAPA Library Quantification Kit according
to the recommended protocol, and then combined to achieve equal final concentrations in two separate pools for
multiplexed sequencing on different flow-cell lanes. The eleven libraries ranged ~11-fold in concentration from 0.67
pM to 7.65 pM, while representation of each index varied between 90% and 127% of expected assigned reads per
lane.