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AMP 2012 HIVposterV3 SM ES CC .pdf


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SENSITIVITY OF HIGH-THROUGHPUT SEQUENCING FOR VIRAL DETECTION IN BLOOD
Erik Samayoa1,2, Samia Naccache1,2, Annie Rein-Weston1,2, Ka-Cheung Luk3, John Hackett, Jr.3, Steve Miller1,2 and Charles Chiu1,2
Viral Diagnostics and Discovery Center, 2Department of Laboratory Medicine, University of California San Francisco, San Francisco CA, USA
and 3Abbott Diagnostics, Abbott Park IL, USA

Background

Results

Unbiased high-throughput sequencing is a promising method for viral
detection in clinical and public health settings. However, optimal
library preparation techniques and limits of detection are largely
unexplored with this technology. We analyzed the effect of various
library preparation protocols on the ability of the Illumina MiSeqTM
instrument to detect viral sequence reads, and performed sensitivity
analysis for detection of human immunodeficiency virus (HIV) in
human plasma.

Approximately 1.3 to 4.3 million sequence reads were obtained for each
sample. At 106 viral copies/mL, pre-nuclease treatment increased viral reads
from 0.03% of total reads (445 reads) with no treatment to 3.1% (45,517
reads). ScriptSeqTM and TruSeqTM-adapted post-DNase protocols generated
comparable percentages of 0.07% (2,928 reads) and 0.08% (1,410 reads) HIV
reads, respectively. At viral loads of 104, 103 and 102 copies/mL pre-nuclease
treatment yielded 2.16% (27,173 reads), 0.35% (3,762 reads) and 0.003% (39
reads), respectively, while pre-DNase treatment yielded 2.23% (28,300 reads),
0.19% (2,020 reads) and 0.009% (50 reads) HIV sequence reads. At viral loads
of 103 and 102 copies/mL the 250-cycle paired-end run yielded 3.35% (52,789
reads) and 0.009% (349 reads) HIV sequence reads. The 3X HIV genome
coverage at 103 and 102 copies/mL was 90.4% and 19.8% respectively for the
250-cycle paired-end run and 48.0% and 7.6% respectively for the 150-cycle
paired-end run. Log-log regression plots of the read percentage against the
spiked HIV titer were linear with an R2 of approximately 0.8703.

Sample Workflow

315 µl
sample
input

Nuclease
Pre-Treatment

Nuclease
Treatment

VDDC MiSeqTM
TruSeqTm-Adapted
Library Prep on
EpmotionTM Robot

Titer
% HIV
(copies/ml) reads

Post-DNase

106

0.094

Pre-DNase

104

1.75

Pre-DNase

103

0.14

Pre-DNase

102

0.0039

Pre-nuclease

104

1.68

Pre-nuclease

103

0.28

Pre-nuclease

102

0.0019

104, 2.2% 104, 2,2%

103, 0.35%

103, 0.19%

-1

106, 0.073%

-1.5
-2

102,

0.0049%

-2.5

102, 0.0029%
-3
0

2

4

6

8

HIV Titer (log scale)
Pre-DNase % Reads

Pre-nuclease % Reads

104
104
103
103
102
102
HIV Titer
(copies/ml)
106
106
106
106
106
104.6

150
150
150
150
150
150

bp
bp
bp
bp
bp
bp

Extraction Kit Used
EZ1 virus 2.0
EZ1 virus 2.0
EZ1
EZ1
EZ1
EZ1
EZ1
EZ1

virus 2.0
virus 2.0
virus 2.0
virus 2.0
virus 2.0
virus 2.0

MiSeq Run
Type
Extraction Kit Used
150 bp
EZ1 virus 2.0
150 bp
EZ1 virus 2.0
150 bp Qiamp UltraSens virus kit
150 bp
EZ1 virus 2.0
150 bp
150 bp

Qiamp UltraSens virus kit
Qiamp UltraSens virus kit

Nuclease Pre- Total R1 Total HIV % HIV Reads
Treatment
Reads
Reads
(10-8)
Pre-DNAse
3,913,494 52,789
1.3%
Pre-DNAse
3,916,365
349
0.0089%

Truseq-Adapted
Truseq-Adapted
Truseq-Adapted
Truseq-Adapted
Truseq-Adapted
Truseq-Adapted

Pre-DNAse
Pre-nuclease*
Pre-DNAse
Pre-nuclease*
Pre-DNAse
Pre-nuclease*

Library Prep
Method
Truseq-Adapted
Truseq-Adapted
Truseq-Adapted
Truseq-Adapted

Nuclease Pre- Total R1 Total HIV % HIV Reads
Treatment
Reads
Reads
(10-10)
Pre-nuclease* 1,437,546 45,517
3.2%
Post-DNase
1,505686
1,417
0.094%
Post-DNase
1,760,695 1,410
0.080%
no treatment 1,392,234
445
0.032%
Post-DNase
Post-DNase

1,280,436
1,262,036
1,081,813
1,093,345
1,063,464
1,415,548

4,031,790
4,342,943

*Pre-nuclease treatment includes DNase, RNase, and benzonase

3.5

106

3.17

Automated
Pre-nuclease TruSeq-Adapted

104

1.68

Automated
Pre-nuclease TruSeq-Adapted

103

0.28

Automated
Pre-nuclease TruSeq-Adapted

102

0.0019

3

R² = 0.8703

2.5
2
1.5
1
0.5
0
-0.5

0

1

2

3

4

5

6

7

HIV Titer (log Scale)

• Manual and automated preps appear substantially equivalent
• Linear increase in % HIV reads with log increase in titer

100
90
80
70
60
50
40
30
20
10
0

150bp vs. 250bp 3X Coverage
Comparison
90.4 %

48.0 %

19.8 %

10.000

150bp vs. 250bp Sensitivity of
Detection Comparison

1.000

0.100

0.010

7.6 %

102

150bp Paired-End Run

Post-DNase % Reads

Library Prep
Method
Truseq-Adapted
Truseq-Adapted

ScriptSeq
ScriptSeq

Manual TruSeqAdapted

103

0.001
0

28,334
27,197
2,029
3,780
52
41

2,928
138

2.2%
2.2%
0.19%
0.35%
0.0049%
0.0029%

0.073%
0.0032%

200

400

600

800

1000

1200

HIV Titer (copies/ml)

HIV Titer (copies/ml)

Table 1 - Data
MiSeq Run
Type
250 bp
250 bp

Manual vs. Automated
Library Prep

% HIV
reads

% HIV Reads

Pre-nuclease

Viral Titers and Sequencing Depth

0
-0.5

Titer
(copies/ml)

Coverage and Sensitivity Comparison

Pre-nuclease and Pre-DNase treatments had comparable yields
and produced a significantly higher percentage of HIV reads than
a Post-DNased sample

Qiagen EZ1TM
Robot
Extraction

Round AB
Amplification

0.5

Library Prep
Method

4

Pre-Treatment Comparison

HIV Titer
(copies/ml)
103
102

ScriptSeqTM
Library Prep

Nuclease
Treatment

% HIV Genome Coverage

Plasma containing HIV at various titers was used to prepare multiplexed
libraries for sequencing on the MiSeq instrument using 300 and 500 cycle
MiSeqTM reagent kits for 150-cycle and 250-cycle paired-end runs. We
assessed two different library preparation methods (Illumina ScriptSeqTM and
TruSeqTM-adapted) as well as nuclease treatment prior to extraction (“prenuclease” / “pre-DNAse”) or immediately after extraction (“post-DNAse”) to
reduce host background contributions to the sequencing reads. Sensitivity
was determined using a dilution series containing 102 – 106 HIV particles
spiked into negative matrix sera. Sequences were identified as viral by
nucleotide BLAST (BLASTn) alignments to HIV (E-value cutoffs of 10-10 and
10-8 were used for 150 base pair (bp) and 250 bp reads, respectively), and the
number of HIV sequence reads generated for each sample as a percentage
of total reads was compared. We compared 3X HIV genome coverage for the
150 and 250 cycle paired-end runs by assembling the BLAST-identified HIV
reads to a reference HIV genome (HIV-1 isolate ES P1261, Genbank
accession number 212674624).

% HIV Sequencing Reads (log scale)

Materials and Methods

Library Preparation Comparison

% HIV Reads

1UCSF-Abbott

250bp Paired-End Run

150bp Paired-End Run

250bp Paired-End Run

Conclusions
• Pre-nuclease / pre-DNase treatment of samples significantly
increased the number and proportion of viral sequence reads in
plasma samples
• Our protocol was able to detect viral sequence reads down to at
least 100 copies/mL
•250bp paired-end runs gave improved coverage and sensitivity
when compared to 150bp paired-end runs
• Sample analysis for viral detection and discovery using these
methods is available as a core laboratory service through the UCSFAbbott Viral Diagnostics and Discovery Center (http://vddc.ucsf.edu)

References and Acknowledgements
Greninger AL, Chen EC, et al. (2010) A metagenomic analysis of pandemic influenza A (2009 H1N1)
infection in patients from North America. PLoS One 5:e13381.
Grard G, Fair J, et al., (2012) A novel rhabdovirus associated with acute hemorrhagic fever in Central
Africa. PloS Pathogens 8(9):e1002924.
Tang P and Chiu CY (2012) Metagenomics for the discovery of novel human viruses 5(2):177-189.
We gratefully acknowledge the support of NIH grants R56-AI08952 and R01-HL105704 and an Abbott
Viral Discovery Award.


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