Covaris Application Note | wwwcovariscom

1

IntroductionAs Next Generation Sequencing (NGS) moves into the clinical applications space requiring higher throughput and sensitivity existing

processes need to be improved to ensure accurate precise data outputs Mechanical DNA shearing using Covarisreg Adaptive Focused

Acousticsreg (AFAreg) technology is recognized as the gold-standard for DNA fragmentation in NGS library preparation AFA hydrodynamic

shear force-based DNA fragmentation is strictly mechanical and isothermal and thus guarantees a highly controlled process while yielding

random unbiased DNA fragment distributions

The Covaris 96 AFA-TUBE TPX Plate is optimized for low DNA input and allows single tube DNA shearing and NGS library preparation This

new engineered polymer vessel incorporates features that effectively control acoustic cavitation to enable reproducible and precisely tuned

hydrodynamic shear forces Its low binding surface guarantees high DNA recovery as well as ease of volume handling down to 10 μl and

picogram DNA inputs Additionally this vessel is fully compatible with magnetic separation procedures thermal cyclers and liquid handling

platforms The AFA-TUBE design is adaptable for scaled-up SBS formats such as 96 384 and higher well densities

In this case study DNA shearing in the AFA-TUBEtrade was paired with Swift Biosciencestrade Accel-NGS 2Sreg Kit for NGS library construction

The 2S kit preserves complexity from low input samples due to enhanced end repair chemistry for highly efficient adapter ligation in a

single tube workflow This combination enables for the first time DNA shearing end repair and adapter ligation in the same vessel thereby

eliminating cumbersome sample transfers We present sequencing data from libraries constructed with this workflow and compare them to

libraries constructed according to standard ldquowith transferrdquo protocol in microTUBE

Combining Covaris 96 AFA-TUBE TPX Plate with Swift Biosciences Accel-NGS 2S Kit for an Optimized Single Tube Workflow for Robust Low Input NGS Library Preparation

Figure 1 Specifications comparison 96 AFA-TUBE TPX Plate vs 96 microTUBE-50 AFA Fiber Plate

96 AFA-TUBE PX Plate 96 microTUBE-50 AFA Plate

NGS Library Construction All steps occur in AFA-TUBE Transfer required for library preparation

Automation Capabilities New plate format allows for use as typical labware Plate holders available for liquid handling platforms

Format Engineered polymer AFA-TUBE Glass-based microTUBE

Sealing Adhesive foil or Cap Map Aluminum adhesive foil

DNA Shearing Volume 10 ndash 50 microl 55 microl

Total Volume Capacity 200 microl 55 microl

Sample Concentration Range Up to 100 ngmicrol Up to 100 ngmicrol

Authors Guillaume Durin1 Elizabeth Weiss1 Jeremy Terry1 Julie Donaldson1 Ulrich Thomann1 Eugenio Daviso1 Jayne Simon1 Gianna Garza1 Justin Lenhart2 Sukhinder Sandhu2 Laurie Kurihara2 and Jim Laugharn1

Affiliation 1 Covaris Inc Woburn MA USA and 2 Swift Biosciences Ann Arbor MI USA

Covaris Application Note | wwwcovariscom

2

Mechanical DNA Shearing PerformancesReproducible DNA Shearing - 48 DNA replicates were treated in the first 6 rows of a 96 AFA-TUBE TPX Plate (1 to 6) for a targeted

size of 150 bp or 350 bp 50 ng human genomic DNA (Promega PN G3041) was sheared on a Covaris LE220-plus with the following

settings

Figure 2 Coupling Accel-NGSreg 2S Plus Library Kit with 96 AFA-TUBE TPX Plate Simplified workflow without transfer step to generate PCR-free NGS libraries

Targeted Size 350 bp 150 bp

PIP (W) 250 250

DF () 20 20

CPB 50 50

Time (s) 52 360

Repair IIEnd Repair amp Polishing

Ligation I3rsquo Ligation of

P7

Ligation II5rsquo Ligation of

P5

DNA Shearing with AFA

Library

Repair IDephosphorylation

Optional PCR varies

27 min

32 min

27 min

22 min

PP

Dephosphorylation at 5rsquo termini prevents chimera formation removal of phosphate at 3rsquo termini improves ligation efficiency

Sequential ligation prevents adapter dimer formation and allows optimized ligation to each strand

Equivalent efficiency at all inputs No adapter titration needed

During Ligation I the P7 adapter is attached to the repaired 3rsquo termini

During Ligation II the P5 adapter is attached to the repaired 5rsquo termini

Optional PCRPCR-free inputs down to 10 ngWith PCR inputs down to 10 pg

1 min96

one

TUBE

-10

Plat

eN

o tr

ansf

er st

eps

Figure 1

100 ng

96 A

FA-T

UBE

TPX

Pla

tes

No

tran

sfer

ste

ps

Covaris Application Note | wwwcovariscom

3

Mechanical DNA Shearing Performances

Figure 4 ndash Reproducible DNA Shearing 48 DNA replicates were treated in the first 6 rows of a 96 oneTUBE-10 AFA Plate (1 to 6) for a targeted size of 150 bp or 350 bp 50 ng human genomic DNA (Promega PN G3041) was sheared on a Covaris LE220-plus with the following settings

Targeted size 350 bp 150 bp

PIP (W) 250 250

DF () 20 20

CPB 50 50

Time (s) 52 360

DNA was analyzed with a Fragment Analyzer (High Sensitivity NGS Fragment Analysis Kit) and the Boxplot graph of the DNA electropherograms was generate by a home-written MATLAB R2016b script

A Overlay of the 48 technical replicates after amplitude normalization 150 bp (Blue) or 350 bp (Red) B Box plot of the 48 replicates for each targeted size (350 bp left 140 bp right) organized by row (1 to 6) and

then position (A to H) Red line is the median blue dot the mean and green diamond the mode Each box represents 25th to 75th region

A

A

B

B

(Figure 3) DNA was analyzed with a Fragment Analyzer (High Sensitivity NGS Fragment Analysis Kit) and the Boxplot graph of the DNA

electropherograms was generated by a custom MATLAB R2016b script

A Overlay of the 48 technical replicates after amplitude normalization 150 bp (Blue) or 350 bp (Red)

B Box plot of the 48 replicates for each targeted size (350 bp left 150 bp right) organized by row (1 to 6) and then position (A to H)

Red line is the median blue dot the mean and green diamond the mode Each box represents 25th to 75th region

----------------- AFA-TUBE 350 bp----------------- AFA-TUBE 150 bp

Reproducible DNA Shearing

Covaris Application Note | wwwcovariscom

4

C

0

50

100

150

200

250

300

350

400

100 50 25 10 5 5 25 1 05 025 01

Aver

age

Mod

e (b

p)

DNA input ngmicroL

Reproducible DNA Shearing

Size (bp)

Figure 5 ndash Dynamic DNA input range Comparison of DNA fragment size distributions at different DNA inputs for a range of 1 ng up to 1 ug Human genomic DNA (Promega PN G3041) was sheared in 96 oneTUBE-10 AFA Plate on a Covaris LE220-plus to a 300bp target size

- A Overlay of 1 replicates per DNA input (1 ng up to 50 ng) DNA was analyzed with a Fragment Analyzer (High Sensitivity NGS Fragment Analysis Kit)

- B Overlay of 1 replicates per DNA input (100 ng up to 1 ug) DNA was analyzed with a Fragment Analyzer (Standard Sensitivity NGS Fragment Analysis Kit)

- C Analysis of average mode size for 8 replicates (entire row) per DNA input across the whole DNA titration range

A

B

C

B

6000

3000

2000

1500

1200

100090

0

800

700

600

500

Size (bp)

400

300

307

305

302

300

200

1001

(1)H1 10 ngmicrol(1)G5 25 ngmicrol(1)F1 50 ngmicrol(1)E2 100 ngmicrol

RFU

2570

2400

2200

2000

1800

1600

1400

1200

1000

800

600

400

200

-24

Figure 5 ndash Dynamic DNA input range Comparison of DNA fragment size distributions at different DNA inputs for a range of 1 ng up to 1 ug Human genomic DNA (Promega PN G3041) was sheared in 96 oneTUBE-10 AFA Plate on a Covaris LE220-plus to a 300bp target size

- A Overlay of 1 replicates per DNA input (1 ng up to 50 ng) DNA was analyzed with a Fragment Analyzer (High Sensitivity NGS Fragment Analysis Kit)

- B Overlay of 1 replicates per DNA input (100 ng up to 1 ug) DNA was analyzed with a Fragment Analyzer (Standard Sensitivity NGS Fragment Analysis Kit)

- C Analysis of average mode size for 8 replicates (entire row) per DNA input across the whole DNA titration range

A

B

C

A(1)B10 01 ngmicrol(1)D1 025 ngmicrol(1)C5 05 ngmicrol(1)B5 1 ngmicrol(1)A6 25 ngmicrol(1)D11 5 ngmicrol

6000

3000

2000

1500

1200

100090

0

800

700

600

500

400

300

302

297

301

292 294301

200

1001

-72

200

400

600

800

1000

1200

RFU

1400

1600

1800

2000

2200

2436

(Figure 4) Comparison of DNA fragment size distributions at different DNA inputs for a range of 1 ng up to 1 μg Human genomic DNA

(Promega PNG3041) was sheared in a 96 AFA-TUBE TPX Plate on a Covaris LE220-plus to a 300 bp target size

A Overlay of 1 replicates per DNA input (1 ng up to 50 ng) DNA was analyzed with a Fragment Analyzer (High Sensitivity NGS

Fragment Analysis Kit)

B Overlay of 1 replicates per DNA input (100 ng up to 1 μg) DNA was analyzed with a Fragment Analyzer (Standard Sensitivity

NGS Fragment Analysis Kit)

C Analysis of average mode size for 6 replicates (entire row) per DNA input across the whole DNA titration range

Covaris Application Note | wwwcovariscom

5

Table 1 Evaluation of sample input and base composition bias

Input Application DNA Covaris Vessel

Mean Fragment

Size (target)Number of Replicates

Mean Fold Coverage

Percent 0X

Coverage

Percent ge30X

Coverage

Percent ge20X

Coverage

100 ng PCR-free

Microbial Mix96 AFA-TUBE

TPX Plate350 bp

4 47x 011 972 986

Microbial Mix microTUBE 4 47x 065 972 986

1 ng9 PCR Cycles

Microbial Mix96 AFA-TUBE

TPX Plate350 bp

4 35x 069 977 988

Microbial Mix microTUBE - - - - -

Figure 5 Picard plots of whole genome sequencing of 100 and 1 ng bacterial DNA mix libraries

100 ng

96 AFA-TUBE TPX Plate

96 oneTUBE-10 AFA Plate microTUBE

100 ng

1 ng

Figure 6

microTUBE

96 oneTUBE-10 AFA Plate microTUBE

100 ng

1 ng

Figure 6

Artificial Microbial Community (AMC) SequencingWe constructed an Artificial Microbial Community (AMC) to study library complexity coverage and bias The AMC is composed of 3 unique

microbial genomes that contain a unique baseline GC composition Each genomic DNA species was pooled with equivalent genome copy

number The AMC genome has a 16565915 bp genome territory

- Escherichia coli (ATCC 10798D-5) 51 GC

- Streptomyces avermitilis (ATCC 31267D-5) 71 GC

- Staphylococcus aureus (ATCC 25923D-5) 33 GC

Libraries were constructed as described in Figure 3 PCR-free libraries from 100 ng and amplified libraries from 1ng of the AMC Low pass

sequencing was carried out on an Illumina MiSeq For each condition two libraries were constructed one in the 96 AFA-TUBE TPX Plate

and one with microTUBE for DNA Shearing and subsequent transfer to PCR plate for library construction (control library)

Covaris Application Note | wwwcovariscom

6

Whole Genome Sequencing on HiSeq X TenPCR-free WGS libraries were constructed as described in Figure 3 using 200 ng Coriell NA12878 genomic DNA For each condition two

libraries were constructed one entirely in the 96 AFA-TUBE TPX Plate and one with microTUBE for DNA Shearing and subsequent transfer

to PCR plate for library construction (control library) Sequencing was carried out by Novogene on the Illumina HiSeq X Ten platform Post

sequencing following exclusion filters were applied before analysis ( exclusion for AFA-TUBE and microTUBE libraries respectively)

- Low mapping quality reads 47 and 47

- Duplicate exclusion 83 and 79

- Low base quality 45 and 42

- Second observation from an insert with overlapping reads 2 and 41

Table 2 WGS PCR-free Libraries

Input Amplification DNA Covaris Vessel Mean Fragment size (target) Number of replicates

200 ng PCR-free NA1287896 AFA-TUBE TPX Plate

350 bp 4microTUBE

Alignment Summary Table

WGS Coverage Table

Table 3 WGS coverage and alignment tables

Mean Coverage

MedianCoverage PCT_5X PCT_10X PCT_15X PCT_20X PCT_25X

96 AFA-TUBE TPX Plate

341 34 981 978 974 965 918

microTUBE 326 33 980 977 973 960 892

Total Reads PF_READS_ALIGNED

PCT_PF_READS _ALIGNED

PF_HQ_ALIGNED _BASES

PF_HQ_ALIGNED _Q20_BASES

PCT_READS_ALIGNED _IN_PAIRS

PCT_ADAPTER_ Dimer

96 AFA-TUBETPX Plate

855785748 854357548 9983 60306787476 58766369045 9886 001

microTUBE 833328788 831857134 9982 58623160032 57180420381 9903 001

Covaris Application Note | wwwcovariscom

7

96 AFA-TUBE TPX Plate

96 AFA-TUBE TPX Plate

microTUBE

microTUBE

Figure 6 Picard plots of 200 ng PCR-free WGS libraries

0 20 40 60 80 100

00

05

10

15

20

All Reads Level All Reads GC Bias Plot

GC of 100 base windows

Frac

tion

of n

orm

aliz

ed c

over

age

010

2030

40

Mea

n ba

se q

ualit

y

minus

Normalized CoverageWindows at GCBase Quality at GC

0 20 40 60 80 100

00

05

10

15

20

All Reads Level All Reads GC Bias Plot

GC of 100 base windows

Frac

tion

of n

orm

aliz

ed c

over

age

010

2030

40

Mea

n ba

se q

ualit

y

minus

Normalized CoverageWindows at GCBase Quality at GC

Figure 7 Cumulative Probability Distributions of Avg Depth of Coverage of the Bad Promoters

Figure 8 Relative ldquoBad Promotersrdquo coverageCharacterizing and measuring bias in sequence data Ross et al Genome Biology 2013 14R51

0102030405060708090

100

Dept

h of

Seq

uenc

ing

Cove

rage

Bad Promoters

Average Coverage oneTUBE Average Coverage microTUBE

Figure 9

AFA-TUBE

Figure 8Bad Promoters List Analysis for WGS DataldquoCharacterizing and measuring bias in sequence datardquo from Ross et al describes a set of 1000 promoters with the lowest relative coverage

(based on an Illumina data set) and called this list the lsquobad promotersrsquo list These ldquoBad Promoters (BP)rdquo are GC-rich (averaging 79 GC

composition)

Covaris Application Note | wwwcovariscom

8

USA Covaris Inc | Tel +1 7819323959 | Fax +1 7819328705 | Email customerservicecovariscomEurope Covaris Ltd | Tel +44 (0)845 872 0100 | Fax +44 (0)845 384 9160 | Email emeacustomerservicecovariscomWeb wwwcovariscom | Applications applicationsupportcovariscom | Service and Support techsupportcovariscomM020072_RevB_Apr2019 | Information subject to change without notice | For research only | Not for use in diagnostic procedures | 2019copy Covaris Inc

Stay Connected

ConclusionCombination of DNA shearing in the AFA-TUBE with Swift Biosciences Accel-NGS 2S Kit enables for the first time DNA shearing end repair

and adapter ligation in the same vessel thereby eliminating cumbersome sample transfers and enabling a streamlined workflow Sequencing

performance metrics in AFA-TUBE are matching or exceeding current benchmark consisting of shearing in microTUBE and then transferring into a

PCR plate for library construction

DNA Shearing with the 96 AFA-TUBE TPX Plate demonstrates robust performances For both conditions analyzed 150 and 350 bp the 48

technical replicates display highly reproducible and tight DNA fragment size distributions Testing performances with different DNA mass also

highlight that as expected with mechanical DNA shearing the protocols are robust across a wide input range

Artificial Microbial Community (AMC) Sequencing data show no adverse biases in coverage uniformity with PCR-free and PCR libraries at inputs

ranging from 1 ng to 100 ng and DNA from species with varying GC

Whole Genome Sequencing on HiSeq X Ten shows an even coverage across the human genome and similar insert size distribution between

AFA-TUBE and microTUBE libraries ldquoBad Promotersrdquo list in depth analysis shows an equivalent coverage between microTUBE and AFA-TUBE

Visit wwwcovariscom and wwwswiftbioscicom for more information

Covaris Application Note | wwwcovariscom

2

Mechanical DNA Shearing PerformancesReproducible DNA Shearing - 48 DNA replicates were treated in the first 6 rows of a 96 AFA-TUBE TPX Plate (1 to 6) for a targeted

size of 150 bp or 350 bp 50 ng human genomic DNA (Promega PN G3041) was sheared on a Covaris LE220-plus with the following

settings

Figure 2 Coupling Accel-NGSreg 2S Plus Library Kit with 96 AFA-TUBE TPX Plate Simplified workflow without transfer step to generate PCR-free NGS libraries

Targeted Size 350 bp 150 bp

PIP (W) 250 250

DF () 20 20

CPB 50 50

Time (s) 52 360

Repair IIEnd Repair amp Polishing

Ligation I3rsquo Ligation of

P7

Ligation II5rsquo Ligation of

P5

DNA Shearing with AFA

Library

Repair IDephosphorylation

Optional PCR varies

27 min

32 min

27 min

22 min

PP

Dephosphorylation at 5rsquo termini prevents chimera formation removal of phosphate at 3rsquo termini improves ligation efficiency

Sequential ligation prevents adapter dimer formation and allows optimized ligation to each strand

Equivalent efficiency at all inputs No adapter titration needed

During Ligation I the P7 adapter is attached to the repaired 3rsquo termini

During Ligation II the P5 adapter is attached to the repaired 5rsquo termini

Optional PCRPCR-free inputs down to 10 ngWith PCR inputs down to 10 pg

1 min96

one

TUBE

-10

Plat

eN

o tr

ansf

er st

eps

Figure 1

100 ng

96 A

FA-T

UBE

TPX

Pla

tes

No

tran

sfer

ste

ps

Covaris Application Note | wwwcovariscom

3

Mechanical DNA Shearing Performances

Figure 4 ndash Reproducible DNA Shearing 48 DNA replicates were treated in the first 6 rows of a 96 oneTUBE-10 AFA Plate (1 to 6) for a targeted size of 150 bp or 350 bp 50 ng human genomic DNA (Promega PN G3041) was sheared on a Covaris LE220-plus with the following settings

Targeted size 350 bp 150 bp

PIP (W) 250 250

DF () 20 20

CPB 50 50

Time (s) 52 360

DNA was analyzed with a Fragment Analyzer (High Sensitivity NGS Fragment Analysis Kit) and the Boxplot graph of the DNA electropherograms was generate by a home-written MATLAB R2016b script

A Overlay of the 48 technical replicates after amplitude normalization 150 bp (Blue) or 350 bp (Red) B Box plot of the 48 replicates for each targeted size (350 bp left 140 bp right) organized by row (1 to 6) and

then position (A to H) Red line is the median blue dot the mean and green diamond the mode Each box represents 25th to 75th region

A

A

B

B

(Figure 3) DNA was analyzed with a Fragment Analyzer (High Sensitivity NGS Fragment Analysis Kit) and the Boxplot graph of the DNA

electropherograms was generated by a custom MATLAB R2016b script

A Overlay of the 48 technical replicates after amplitude normalization 150 bp (Blue) or 350 bp (Red)

B Box plot of the 48 replicates for each targeted size (350 bp left 150 bp right) organized by row (1 to 6) and then position (A to H)

Red line is the median blue dot the mean and green diamond the mode Each box represents 25th to 75th region

----------------- AFA-TUBE 350 bp----------------- AFA-TUBE 150 bp

Reproducible DNA Shearing

Covaris Application Note | wwwcovariscom

4

C

0

50

100

150

200

250

300

350

400

100 50 25 10 5 5 25 1 05 025 01

Aver

age

Mod

e (b

p)

DNA input ngmicroL

Reproducible DNA Shearing

Size (bp)

Figure 5 ndash Dynamic DNA input range Comparison of DNA fragment size distributions at different DNA inputs for a range of 1 ng up to 1 ug Human genomic DNA (Promega PN G3041) was sheared in 96 oneTUBE-10 AFA Plate on a Covaris LE220-plus to a 300bp target size

- A Overlay of 1 replicates per DNA input (1 ng up to 50 ng) DNA was analyzed with a Fragment Analyzer (High Sensitivity NGS Fragment Analysis Kit)

- B Overlay of 1 replicates per DNA input (100 ng up to 1 ug) DNA was analyzed with a Fragment Analyzer (Standard Sensitivity NGS Fragment Analysis Kit)

- C Analysis of average mode size for 8 replicates (entire row) per DNA input across the whole DNA titration range

A

B

C

B

6000

3000

2000

1500

1200

100090

0

800

700

600

500

Size (bp)

400

300

307

305

302

300

200

1001

(1)H1 10 ngmicrol(1)G5 25 ngmicrol(1)F1 50 ngmicrol(1)E2 100 ngmicrol

RFU

2570

2400

2200

2000

1800

1600

1400

1200

1000

800

600

400

200

-24

Figure 5 ndash Dynamic DNA input range Comparison of DNA fragment size distributions at different DNA inputs for a range of 1 ng up to 1 ug Human genomic DNA (Promega PN G3041) was sheared in 96 oneTUBE-10 AFA Plate on a Covaris LE220-plus to a 300bp target size

- A Overlay of 1 replicates per DNA input (1 ng up to 50 ng) DNA was analyzed with a Fragment Analyzer (High Sensitivity NGS Fragment Analysis Kit)

- B Overlay of 1 replicates per DNA input (100 ng up to 1 ug) DNA was analyzed with a Fragment Analyzer (Standard Sensitivity NGS Fragment Analysis Kit)

- C Analysis of average mode size for 8 replicates (entire row) per DNA input across the whole DNA titration range

A

B

C

A(1)B10 01 ngmicrol(1)D1 025 ngmicrol(1)C5 05 ngmicrol(1)B5 1 ngmicrol(1)A6 25 ngmicrol(1)D11 5 ngmicrol

6000

3000

2000

1500

1200

100090

0

800

700

600

500

400

300

302

297

301

292 294301

200

1001

-72

200

400

600

800

1000

1200

RFU

1400

1600

1800

2000

2200

2436

(Figure 4) Comparison of DNA fragment size distributions at different DNA inputs for a range of 1 ng up to 1 μg Human genomic DNA

(Promega PNG3041) was sheared in a 96 AFA-TUBE TPX Plate on a Covaris LE220-plus to a 300 bp target size

A Overlay of 1 replicates per DNA input (1 ng up to 50 ng) DNA was analyzed with a Fragment Analyzer (High Sensitivity NGS

Fragment Analysis Kit)

B Overlay of 1 replicates per DNA input (100 ng up to 1 μg) DNA was analyzed with a Fragment Analyzer (Standard Sensitivity

NGS Fragment Analysis Kit)

C Analysis of average mode size for 6 replicates (entire row) per DNA input across the whole DNA titration range

Covaris Application Note | wwwcovariscom

5

Table 1 Evaluation of sample input and base composition bias

Input Application DNA Covaris Vessel

Mean Fragment

Size (target)Number of Replicates

Mean Fold Coverage

Percent 0X

Coverage

Percent ge30X

Coverage

Percent ge20X

Coverage

100 ng PCR-free

Microbial Mix96 AFA-TUBE

TPX Plate350 bp

4 47x 011 972 986

Microbial Mix microTUBE 4 47x 065 972 986

1 ng9 PCR Cycles

Microbial Mix96 AFA-TUBE

TPX Plate350 bp

4 35x 069 977 988

Microbial Mix microTUBE - - - - -

Figure 5 Picard plots of whole genome sequencing of 100 and 1 ng bacterial DNA mix libraries

100 ng

96 AFA-TUBE TPX Plate

96 oneTUBE-10 AFA Plate microTUBE

100 ng

1 ng

Figure 6

microTUBE

96 oneTUBE-10 AFA Plate microTUBE

100 ng

1 ng

Figure 6

Artificial Microbial Community (AMC) SequencingWe constructed an Artificial Microbial Community (AMC) to study library complexity coverage and bias The AMC is composed of 3 unique

microbial genomes that contain a unique baseline GC composition Each genomic DNA species was pooled with equivalent genome copy

number The AMC genome has a 16565915 bp genome territory

- Escherichia coli (ATCC 10798D-5) 51 GC

- Streptomyces avermitilis (ATCC 31267D-5) 71 GC

- Staphylococcus aureus (ATCC 25923D-5) 33 GC

Libraries were constructed as described in Figure 3 PCR-free libraries from 100 ng and amplified libraries from 1ng of the AMC Low pass

sequencing was carried out on an Illumina MiSeq For each condition two libraries were constructed one in the 96 AFA-TUBE TPX Plate

and one with microTUBE for DNA Shearing and subsequent transfer to PCR plate for library construction (control library)

Covaris Application Note | wwwcovariscom

6

Whole Genome Sequencing on HiSeq X TenPCR-free WGS libraries were constructed as described in Figure 3 using 200 ng Coriell NA12878 genomic DNA For each condition two

libraries were constructed one entirely in the 96 AFA-TUBE TPX Plate and one with microTUBE for DNA Shearing and subsequent transfer

to PCR plate for library construction (control library) Sequencing was carried out by Novogene on the Illumina HiSeq X Ten platform Post

sequencing following exclusion filters were applied before analysis ( exclusion for AFA-TUBE and microTUBE libraries respectively)

- Low mapping quality reads 47 and 47

- Duplicate exclusion 83 and 79

- Low base quality 45 and 42

- Second observation from an insert with overlapping reads 2 and 41

Table 2 WGS PCR-free Libraries

Input Amplification DNA Covaris Vessel Mean Fragment size (target) Number of replicates

200 ng PCR-free NA1287896 AFA-TUBE TPX Plate

350 bp 4microTUBE

Alignment Summary Table

WGS Coverage Table

Table 3 WGS coverage and alignment tables

Mean Coverage

MedianCoverage PCT_5X PCT_10X PCT_15X PCT_20X PCT_25X

96 AFA-TUBE TPX Plate

341 34 981 978 974 965 918

microTUBE 326 33 980 977 973 960 892

Total Reads PF_READS_ALIGNED

PCT_PF_READS _ALIGNED

PF_HQ_ALIGNED _BASES

PF_HQ_ALIGNED _Q20_BASES

PCT_READS_ALIGNED _IN_PAIRS

PCT_ADAPTER_ Dimer

96 AFA-TUBETPX Plate

855785748 854357548 9983 60306787476 58766369045 9886 001

microTUBE 833328788 831857134 9982 58623160032 57180420381 9903 001

Covaris Application Note | wwwcovariscom

7

96 AFA-TUBE TPX Plate

96 AFA-TUBE TPX Plate

microTUBE

microTUBE

Figure 6 Picard plots of 200 ng PCR-free WGS libraries

0 20 40 60 80 100

00

05

10

15

20

All Reads Level All Reads GC Bias Plot

GC of 100 base windows

Frac

tion

of n

orm

aliz

ed c

over

age

010

2030

40

Mea

n ba

se q

ualit

y

minus

Normalized CoverageWindows at GCBase Quality at GC

0 20 40 60 80 100

00

05

10

15

20

All Reads Level All Reads GC Bias Plot

GC of 100 base windows

Frac

tion

of n

orm

aliz

ed c

over

age

010

2030

40

Mea

n ba

se q

ualit

y

minus

Normalized CoverageWindows at GCBase Quality at GC

Figure 7 Cumulative Probability Distributions of Avg Depth of Coverage of the Bad Promoters

Figure 8 Relative ldquoBad Promotersrdquo coverageCharacterizing and measuring bias in sequence data Ross et al Genome Biology 2013 14R51

0102030405060708090

100

Dept

h of

Seq

uenc

ing

Cove

rage

Bad Promoters

Average Coverage oneTUBE Average Coverage microTUBE

Figure 9

AFA-TUBE

Figure 8Bad Promoters List Analysis for WGS DataldquoCharacterizing and measuring bias in sequence datardquo from Ross et al describes a set of 1000 promoters with the lowest relative coverage

(based on an Illumina data set) and called this list the lsquobad promotersrsquo list These ldquoBad Promoters (BP)rdquo are GC-rich (averaging 79 GC

composition)

Covaris Application Note | wwwcovariscom

8

USA Covaris Inc | Tel +1 7819323959 | Fax +1 7819328705 | Email customerservicecovariscomEurope Covaris Ltd | Tel +44 (0)845 872 0100 | Fax +44 (0)845 384 9160 | Email emeacustomerservicecovariscomWeb wwwcovariscom | Applications applicationsupportcovariscom | Service and Support techsupportcovariscomM020072_RevB_Apr2019 | Information subject to change without notice | For research only | Not for use in diagnostic procedures | 2019copy Covaris Inc

Stay Connected

ConclusionCombination of DNA shearing in the AFA-TUBE with Swift Biosciences Accel-NGS 2S Kit enables for the first time DNA shearing end repair

and adapter ligation in the same vessel thereby eliminating cumbersome sample transfers and enabling a streamlined workflow Sequencing

performance metrics in AFA-TUBE are matching or exceeding current benchmark consisting of shearing in microTUBE and then transferring into a

PCR plate for library construction

DNA Shearing with the 96 AFA-TUBE TPX Plate demonstrates robust performances For both conditions analyzed 150 and 350 bp the 48

technical replicates display highly reproducible and tight DNA fragment size distributions Testing performances with different DNA mass also

highlight that as expected with mechanical DNA shearing the protocols are robust across a wide input range

Artificial Microbial Community (AMC) Sequencing data show no adverse biases in coverage uniformity with PCR-free and PCR libraries at inputs

ranging from 1 ng to 100 ng and DNA from species with varying GC

Whole Genome Sequencing on HiSeq X Ten shows an even coverage across the human genome and similar insert size distribution between

AFA-TUBE and microTUBE libraries ldquoBad Promotersrdquo list in depth analysis shows an equivalent coverage between microTUBE and AFA-TUBE

Visit wwwcovariscom and wwwswiftbioscicom for more information

Covaris Application Note | wwwcovariscom

3

Mechanical DNA Shearing Performances

Figure 4 ndash Reproducible DNA Shearing 48 DNA replicates were treated in the first 6 rows of a 96 oneTUBE-10 AFA Plate (1 to 6) for a targeted size of 150 bp or 350 bp 50 ng human genomic DNA (Promega PN G3041) was sheared on a Covaris LE220-plus with the following settings

Targeted size 350 bp 150 bp

PIP (W) 250 250

DF () 20 20

CPB 50 50

Time (s) 52 360

DNA was analyzed with a Fragment Analyzer (High Sensitivity NGS Fragment Analysis Kit) and the Boxplot graph of the DNA electropherograms was generate by a home-written MATLAB R2016b script

A Overlay of the 48 technical replicates after amplitude normalization 150 bp (Blue) or 350 bp (Red) B Box plot of the 48 replicates for each targeted size (350 bp left 140 bp right) organized by row (1 to 6) and

then position (A to H) Red line is the median blue dot the mean and green diamond the mode Each box represents 25th to 75th region

A

A

B

B

(Figure 3) DNA was analyzed with a Fragment Analyzer (High Sensitivity NGS Fragment Analysis Kit) and the Boxplot graph of the DNA

electropherograms was generated by a custom MATLAB R2016b script

A Overlay of the 48 technical replicates after amplitude normalization 150 bp (Blue) or 350 bp (Red)

B Box plot of the 48 replicates for each targeted size (350 bp left 150 bp right) organized by row (1 to 6) and then position (A to H)

Red line is the median blue dot the mean and green diamond the mode Each box represents 25th to 75th region

----------------- AFA-TUBE 350 bp----------------- AFA-TUBE 150 bp

Reproducible DNA Shearing

Covaris Application Note | wwwcovariscom

4

C

0

50

100

150

200

250

300

350

400

100 50 25 10 5 5 25 1 05 025 01

Aver

age

Mod

e (b

p)

DNA input ngmicroL

Reproducible DNA Shearing

Size (bp)

Figure 5 ndash Dynamic DNA input range Comparison of DNA fragment size distributions at different DNA inputs for a range of 1 ng up to 1 ug Human genomic DNA (Promega PN G3041) was sheared in 96 oneTUBE-10 AFA Plate on a Covaris LE220-plus to a 300bp target size

- A Overlay of 1 replicates per DNA input (1 ng up to 50 ng) DNA was analyzed with a Fragment Analyzer (High Sensitivity NGS Fragment Analysis Kit)

- B Overlay of 1 replicates per DNA input (100 ng up to 1 ug) DNA was analyzed with a Fragment Analyzer (Standard Sensitivity NGS Fragment Analysis Kit)

- C Analysis of average mode size for 8 replicates (entire row) per DNA input across the whole DNA titration range

A

B

C

B

6000

3000

2000

1500

1200

100090

0

800

700

600

500

Size (bp)

400

300

307

305

302

300

200

1001

(1)H1 10 ngmicrol(1)G5 25 ngmicrol(1)F1 50 ngmicrol(1)E2 100 ngmicrol

RFU

2570

2400

2200

2000

1800

1600

1400

1200

1000

800

600

400

200

-24

Figure 5 ndash Dynamic DNA input range Comparison of DNA fragment size distributions at different DNA inputs for a range of 1 ng up to 1 ug Human genomic DNA (Promega PN G3041) was sheared in 96 oneTUBE-10 AFA Plate on a Covaris LE220-plus to a 300bp target size

- A Overlay of 1 replicates per DNA input (1 ng up to 50 ng) DNA was analyzed with a Fragment Analyzer (High Sensitivity NGS Fragment Analysis Kit)

- B Overlay of 1 replicates per DNA input (100 ng up to 1 ug) DNA was analyzed with a Fragment Analyzer (Standard Sensitivity NGS Fragment Analysis Kit)

- C Analysis of average mode size for 8 replicates (entire row) per DNA input across the whole DNA titration range

A

B

C

A(1)B10 01 ngmicrol(1)D1 025 ngmicrol(1)C5 05 ngmicrol(1)B5 1 ngmicrol(1)A6 25 ngmicrol(1)D11 5 ngmicrol

6000

3000

2000

1500

1200

100090

0

800

700

600

500

400

300

302

297

301

292 294301

200

1001

-72

200

400

600

800

1000

1200

RFU

1400

1600

1800

2000

2200

2436

(Figure 4) Comparison of DNA fragment size distributions at different DNA inputs for a range of 1 ng up to 1 μg Human genomic DNA

(Promega PNG3041) was sheared in a 96 AFA-TUBE TPX Plate on a Covaris LE220-plus to a 300 bp target size

A Overlay of 1 replicates per DNA input (1 ng up to 50 ng) DNA was analyzed with a Fragment Analyzer (High Sensitivity NGS

Fragment Analysis Kit)

B Overlay of 1 replicates per DNA input (100 ng up to 1 μg) DNA was analyzed with a Fragment Analyzer (Standard Sensitivity

NGS Fragment Analysis Kit)

C Analysis of average mode size for 6 replicates (entire row) per DNA input across the whole DNA titration range

Covaris Application Note | wwwcovariscom

5

Table 1 Evaluation of sample input and base composition bias

Input Application DNA Covaris Vessel

Mean Fragment

Size (target)Number of Replicates

Mean Fold Coverage

Percent 0X

Coverage

Percent ge30X

Coverage

Percent ge20X

Coverage

100 ng PCR-free

Microbial Mix96 AFA-TUBE

TPX Plate350 bp

4 47x 011 972 986

Microbial Mix microTUBE 4 47x 065 972 986

1 ng9 PCR Cycles

Microbial Mix96 AFA-TUBE

TPX Plate350 bp

4 35x 069 977 988

Microbial Mix microTUBE - - - - -

Figure 5 Picard plots of whole genome sequencing of 100 and 1 ng bacterial DNA mix libraries

100 ng

96 AFA-TUBE TPX Plate

96 oneTUBE-10 AFA Plate microTUBE

100 ng

1 ng

Figure 6

microTUBE

96 oneTUBE-10 AFA Plate microTUBE

100 ng

1 ng

Figure 6

Artificial Microbial Community (AMC) SequencingWe constructed an Artificial Microbial Community (AMC) to study library complexity coverage and bias The AMC is composed of 3 unique

microbial genomes that contain a unique baseline GC composition Each genomic DNA species was pooled with equivalent genome copy

number The AMC genome has a 16565915 bp genome territory

- Escherichia coli (ATCC 10798D-5) 51 GC

- Streptomyces avermitilis (ATCC 31267D-5) 71 GC

- Staphylococcus aureus (ATCC 25923D-5) 33 GC

Libraries were constructed as described in Figure 3 PCR-free libraries from 100 ng and amplified libraries from 1ng of the AMC Low pass

sequencing was carried out on an Illumina MiSeq For each condition two libraries were constructed one in the 96 AFA-TUBE TPX Plate

and one with microTUBE for DNA Shearing and subsequent transfer to PCR plate for library construction (control library)

Covaris Application Note | wwwcovariscom

6

Whole Genome Sequencing on HiSeq X TenPCR-free WGS libraries were constructed as described in Figure 3 using 200 ng Coriell NA12878 genomic DNA For each condition two

libraries were constructed one entirely in the 96 AFA-TUBE TPX Plate and one with microTUBE for DNA Shearing and subsequent transfer

to PCR plate for library construction (control library) Sequencing was carried out by Novogene on the Illumina HiSeq X Ten platform Post

sequencing following exclusion filters were applied before analysis ( exclusion for AFA-TUBE and microTUBE libraries respectively)

- Low mapping quality reads 47 and 47

- Duplicate exclusion 83 and 79

- Low base quality 45 and 42

- Second observation from an insert with overlapping reads 2 and 41

Table 2 WGS PCR-free Libraries

Input Amplification DNA Covaris Vessel Mean Fragment size (target) Number of replicates

200 ng PCR-free NA1287896 AFA-TUBE TPX Plate

350 bp 4microTUBE

Alignment Summary Table

WGS Coverage Table

Table 3 WGS coverage and alignment tables

Mean Coverage

MedianCoverage PCT_5X PCT_10X PCT_15X PCT_20X PCT_25X

96 AFA-TUBE TPX Plate

341 34 981 978 974 965 918

microTUBE 326 33 980 977 973 960 892

Total Reads PF_READS_ALIGNED

PCT_PF_READS _ALIGNED

PF_HQ_ALIGNED _BASES

PF_HQ_ALIGNED _Q20_BASES

PCT_READS_ALIGNED _IN_PAIRS

PCT_ADAPTER_ Dimer

96 AFA-TUBETPX Plate

855785748 854357548 9983 60306787476 58766369045 9886 001

microTUBE 833328788 831857134 9982 58623160032 57180420381 9903 001

Covaris Application Note | wwwcovariscom

7

96 AFA-TUBE TPX Plate

96 AFA-TUBE TPX Plate

microTUBE

microTUBE

Figure 6 Picard plots of 200 ng PCR-free WGS libraries

0 20 40 60 80 100

00

05

10

15

20

All Reads Level All Reads GC Bias Plot

GC of 100 base windows

Frac

tion

of n

orm

aliz

ed c

over

age

010

2030

40

Mea

n ba

se q

ualit

y

minus

Normalized CoverageWindows at GCBase Quality at GC

0 20 40 60 80 100

00

05

10

15

20

All Reads Level All Reads GC Bias Plot

GC of 100 base windows

Frac

tion

of n

orm

aliz

ed c

over

age

010

2030

40

Mea

n ba

se q

ualit

y

minus

Normalized CoverageWindows at GCBase Quality at GC

Figure 7 Cumulative Probability Distributions of Avg Depth of Coverage of the Bad Promoters

Figure 8 Relative ldquoBad Promotersrdquo coverageCharacterizing and measuring bias in sequence data Ross et al Genome Biology 2013 14R51

0102030405060708090

100

Dept

h of

Seq

uenc

ing

Cove

rage

Bad Promoters

Average Coverage oneTUBE Average Coverage microTUBE

Figure 9

AFA-TUBE

Figure 8Bad Promoters List Analysis for WGS DataldquoCharacterizing and measuring bias in sequence datardquo from Ross et al describes a set of 1000 promoters with the lowest relative coverage

(based on an Illumina data set) and called this list the lsquobad promotersrsquo list These ldquoBad Promoters (BP)rdquo are GC-rich (averaging 79 GC

composition)

Covaris Application Note | wwwcovariscom

8

USA Covaris Inc | Tel +1 7819323959 | Fax +1 7819328705 | Email customerservicecovariscomEurope Covaris Ltd | Tel +44 (0)845 872 0100 | Fax +44 (0)845 384 9160 | Email emeacustomerservicecovariscomWeb wwwcovariscom | Applications applicationsupportcovariscom | Service and Support techsupportcovariscomM020072_RevB_Apr2019 | Information subject to change without notice | For research only | Not for use in diagnostic procedures | 2019copy Covaris Inc

Stay Connected

ConclusionCombination of DNA shearing in the AFA-TUBE with Swift Biosciences Accel-NGS 2S Kit enables for the first time DNA shearing end repair

and adapter ligation in the same vessel thereby eliminating cumbersome sample transfers and enabling a streamlined workflow Sequencing

performance metrics in AFA-TUBE are matching or exceeding current benchmark consisting of shearing in microTUBE and then transferring into a

PCR plate for library construction

DNA Shearing with the 96 AFA-TUBE TPX Plate demonstrates robust performances For both conditions analyzed 150 and 350 bp the 48

technical replicates display highly reproducible and tight DNA fragment size distributions Testing performances with different DNA mass also

highlight that as expected with mechanical DNA shearing the protocols are robust across a wide input range

Artificial Microbial Community (AMC) Sequencing data show no adverse biases in coverage uniformity with PCR-free and PCR libraries at inputs

ranging from 1 ng to 100 ng and DNA from species with varying GC

Whole Genome Sequencing on HiSeq X Ten shows an even coverage across the human genome and similar insert size distribution between

AFA-TUBE and microTUBE libraries ldquoBad Promotersrdquo list in depth analysis shows an equivalent coverage between microTUBE and AFA-TUBE

Visit wwwcovariscom and wwwswiftbioscicom for more information

Covaris Application Note | wwwcovariscom

4

C

0

50

100

150

200

250

300

350

400

100 50 25 10 5 5 25 1 05 025 01

Aver

age

Mod

e (b

p)

DNA input ngmicroL

Reproducible DNA Shearing

Size (bp)

Figure 5 ndash Dynamic DNA input range Comparison of DNA fragment size distributions at different DNA inputs for a range of 1 ng up to 1 ug Human genomic DNA (Promega PN G3041) was sheared in 96 oneTUBE-10 AFA Plate on a Covaris LE220-plus to a 300bp target size

- A Overlay of 1 replicates per DNA input (1 ng up to 50 ng) DNA was analyzed with a Fragment Analyzer (High Sensitivity NGS Fragment Analysis Kit)

- B Overlay of 1 replicates per DNA input (100 ng up to 1 ug) DNA was analyzed with a Fragment Analyzer (Standard Sensitivity NGS Fragment Analysis Kit)

- C Analysis of average mode size for 8 replicates (entire row) per DNA input across the whole DNA titration range

A

B

C

B

6000

3000

2000

1500

1200

100090

0

800

700

600

500

Size (bp)

400

300

307

305

302

300

200

1001

(1)H1 10 ngmicrol(1)G5 25 ngmicrol(1)F1 50 ngmicrol(1)E2 100 ngmicrol

RFU

2570

2400

2200

2000

1800

1600

1400

1200

1000

800

600

400

200

-24

Figure 5 ndash Dynamic DNA input range Comparison of DNA fragment size distributions at different DNA inputs for a range of 1 ng up to 1 ug Human genomic DNA (Promega PN G3041) was sheared in 96 oneTUBE-10 AFA Plate on a Covaris LE220-plus to a 300bp target size

- A Overlay of 1 replicates per DNA input (1 ng up to 50 ng) DNA was analyzed with a Fragment Analyzer (High Sensitivity NGS Fragment Analysis Kit)

- B Overlay of 1 replicates per DNA input (100 ng up to 1 ug) DNA was analyzed with a Fragment Analyzer (Standard Sensitivity NGS Fragment Analysis Kit)

- C Analysis of average mode size for 8 replicates (entire row) per DNA input across the whole DNA titration range

A

B

C

A(1)B10 01 ngmicrol(1)D1 025 ngmicrol(1)C5 05 ngmicrol(1)B5 1 ngmicrol(1)A6 25 ngmicrol(1)D11 5 ngmicrol

6000

3000

2000

1500

1200

100090

0

800

700

600

500

400

300

302

297

301

292 294301

200

1001

-72

200

400

600

800

1000

1200

RFU

1400

1600

1800

2000

2200

2436

(Figure 4) Comparison of DNA fragment size distributions at different DNA inputs for a range of 1 ng up to 1 μg Human genomic DNA

(Promega PNG3041) was sheared in a 96 AFA-TUBE TPX Plate on a Covaris LE220-plus to a 300 bp target size

A Overlay of 1 replicates per DNA input (1 ng up to 50 ng) DNA was analyzed with a Fragment Analyzer (High Sensitivity NGS

Fragment Analysis Kit)

B Overlay of 1 replicates per DNA input (100 ng up to 1 μg) DNA was analyzed with a Fragment Analyzer (Standard Sensitivity

NGS Fragment Analysis Kit)

C Analysis of average mode size for 6 replicates (entire row) per DNA input across the whole DNA titration range

Covaris Application Note | wwwcovariscom

5

Table 1 Evaluation of sample input and base composition bias

Input Application DNA Covaris Vessel

Mean Fragment

Size (target)Number of Replicates

Mean Fold Coverage

Percent 0X

Coverage

Percent ge30X

Coverage

Percent ge20X

Coverage

100 ng PCR-free

Microbial Mix96 AFA-TUBE

TPX Plate350 bp

4 47x 011 972 986

Microbial Mix microTUBE 4 47x 065 972 986

1 ng9 PCR Cycles

Microbial Mix96 AFA-TUBE

TPX Plate350 bp

4 35x 069 977 988

Microbial Mix microTUBE - - - - -

Figure 5 Picard plots of whole genome sequencing of 100 and 1 ng bacterial DNA mix libraries

100 ng

96 AFA-TUBE TPX Plate

96 oneTUBE-10 AFA Plate microTUBE

100 ng

1 ng

Figure 6

microTUBE

96 oneTUBE-10 AFA Plate microTUBE

100 ng

1 ng

Figure 6

Artificial Microbial Community (AMC) SequencingWe constructed an Artificial Microbial Community (AMC) to study library complexity coverage and bias The AMC is composed of 3 unique

microbial genomes that contain a unique baseline GC composition Each genomic DNA species was pooled with equivalent genome copy

number The AMC genome has a 16565915 bp genome territory

- Escherichia coli (ATCC 10798D-5) 51 GC

- Streptomyces avermitilis (ATCC 31267D-5) 71 GC

- Staphylococcus aureus (ATCC 25923D-5) 33 GC

Libraries were constructed as described in Figure 3 PCR-free libraries from 100 ng and amplified libraries from 1ng of the AMC Low pass

sequencing was carried out on an Illumina MiSeq For each condition two libraries were constructed one in the 96 AFA-TUBE TPX Plate

and one with microTUBE for DNA Shearing and subsequent transfer to PCR plate for library construction (control library)

Covaris Application Note | wwwcovariscom

6

Whole Genome Sequencing on HiSeq X TenPCR-free WGS libraries were constructed as described in Figure 3 using 200 ng Coriell NA12878 genomic DNA For each condition two

libraries were constructed one entirely in the 96 AFA-TUBE TPX Plate and one with microTUBE for DNA Shearing and subsequent transfer

to PCR plate for library construction (control library) Sequencing was carried out by Novogene on the Illumina HiSeq X Ten platform Post

sequencing following exclusion filters were applied before analysis ( exclusion for AFA-TUBE and microTUBE libraries respectively)

- Low mapping quality reads 47 and 47

- Duplicate exclusion 83 and 79

- Low base quality 45 and 42

- Second observation from an insert with overlapping reads 2 and 41

Table 2 WGS PCR-free Libraries

Input Amplification DNA Covaris Vessel Mean Fragment size (target) Number of replicates

200 ng PCR-free NA1287896 AFA-TUBE TPX Plate

350 bp 4microTUBE

Alignment Summary Table

WGS Coverage Table

Table 3 WGS coverage and alignment tables

Mean Coverage

MedianCoverage PCT_5X PCT_10X PCT_15X PCT_20X PCT_25X

96 AFA-TUBE TPX Plate

341 34 981 978 974 965 918

microTUBE 326 33 980 977 973 960 892

Total Reads PF_READS_ALIGNED

PCT_PF_READS _ALIGNED

PF_HQ_ALIGNED _BASES

PF_HQ_ALIGNED _Q20_BASES

PCT_READS_ALIGNED _IN_PAIRS

PCT_ADAPTER_ Dimer

96 AFA-TUBETPX Plate

855785748 854357548 9983 60306787476 58766369045 9886 001

microTUBE 833328788 831857134 9982 58623160032 57180420381 9903 001

Covaris Application Note | wwwcovariscom

7

96 AFA-TUBE TPX Plate

96 AFA-TUBE TPX Plate

microTUBE

microTUBE

Figure 6 Picard plots of 200 ng PCR-free WGS libraries

0 20 40 60 80 100

00

05

10

15

20

All Reads Level All Reads GC Bias Plot

GC of 100 base windows

Frac

tion

of n

orm

aliz

ed c

over

age

010

2030

40

Mea

n ba

se q

ualit

y

minus

Normalized CoverageWindows at GCBase Quality at GC

0 20 40 60 80 100

00

05

10

15

20

All Reads Level All Reads GC Bias Plot

GC of 100 base windows

Frac

tion

of n

orm

aliz

ed c

over

age

010

2030

40

Mea

n ba

se q

ualit

y

minus

Normalized CoverageWindows at GCBase Quality at GC

Figure 7 Cumulative Probability Distributions of Avg Depth of Coverage of the Bad Promoters

Figure 8 Relative ldquoBad Promotersrdquo coverageCharacterizing and measuring bias in sequence data Ross et al Genome Biology 2013 14R51

0102030405060708090

100

Dept

h of

Seq

uenc

ing

Cove

rage

Bad Promoters

Average Coverage oneTUBE Average Coverage microTUBE

Figure 9

AFA-TUBE

Figure 8Bad Promoters List Analysis for WGS DataldquoCharacterizing and measuring bias in sequence datardquo from Ross et al describes a set of 1000 promoters with the lowest relative coverage

(based on an Illumina data set) and called this list the lsquobad promotersrsquo list These ldquoBad Promoters (BP)rdquo are GC-rich (averaging 79 GC

composition)

Covaris Application Note | wwwcovariscom

8

USA Covaris Inc | Tel +1 7819323959 | Fax +1 7819328705 | Email customerservicecovariscomEurope Covaris Ltd | Tel +44 (0)845 872 0100 | Fax +44 (0)845 384 9160 | Email emeacustomerservicecovariscomWeb wwwcovariscom | Applications applicationsupportcovariscom | Service and Support techsupportcovariscomM020072_RevB_Apr2019 | Information subject to change without notice | For research only | Not for use in diagnostic procedures | 2019copy Covaris Inc

Stay Connected

ConclusionCombination of DNA shearing in the AFA-TUBE with Swift Biosciences Accel-NGS 2S Kit enables for the first time DNA shearing end repair

and adapter ligation in the same vessel thereby eliminating cumbersome sample transfers and enabling a streamlined workflow Sequencing

performance metrics in AFA-TUBE are matching or exceeding current benchmark consisting of shearing in microTUBE and then transferring into a

PCR plate for library construction

DNA Shearing with the 96 AFA-TUBE TPX Plate demonstrates robust performances For both conditions analyzed 150 and 350 bp the 48

technical replicates display highly reproducible and tight DNA fragment size distributions Testing performances with different DNA mass also

highlight that as expected with mechanical DNA shearing the protocols are robust across a wide input range

Artificial Microbial Community (AMC) Sequencing data show no adverse biases in coverage uniformity with PCR-free and PCR libraries at inputs

ranging from 1 ng to 100 ng and DNA from species with varying GC

Whole Genome Sequencing on HiSeq X Ten shows an even coverage across the human genome and similar insert size distribution between

AFA-TUBE and microTUBE libraries ldquoBad Promotersrdquo list in depth analysis shows an equivalent coverage between microTUBE and AFA-TUBE

Visit wwwcovariscom and wwwswiftbioscicom for more information

Covaris Application Note | wwwcovariscom

5

Table 1 Evaluation of sample input and base composition bias

Input Application DNA Covaris Vessel

Mean Fragment

Size (target)Number of Replicates

Mean Fold Coverage

Percent 0X

Coverage

Percent ge30X

Coverage

Percent ge20X

Coverage

100 ng PCR-free

Microbial Mix96 AFA-TUBE

TPX Plate350 bp

4 47x 011 972 986

Microbial Mix microTUBE 4 47x 065 972 986

1 ng9 PCR Cycles

Microbial Mix96 AFA-TUBE

TPX Plate350 bp

4 35x 069 977 988

Microbial Mix microTUBE - - - - -

Figure 5 Picard plots of whole genome sequencing of 100 and 1 ng bacterial DNA mix libraries

100 ng

96 AFA-TUBE TPX Plate

96 oneTUBE-10 AFA Plate microTUBE

100 ng

1 ng

Figure 6

microTUBE

96 oneTUBE-10 AFA Plate microTUBE

100 ng

1 ng

Figure 6

Artificial Microbial Community (AMC) SequencingWe constructed an Artificial Microbial Community (AMC) to study library complexity coverage and bias The AMC is composed of 3 unique

microbial genomes that contain a unique baseline GC composition Each genomic DNA species was pooled with equivalent genome copy

number The AMC genome has a 16565915 bp genome territory

- Escherichia coli (ATCC 10798D-5) 51 GC

- Streptomyces avermitilis (ATCC 31267D-5) 71 GC

- Staphylococcus aureus (ATCC 25923D-5) 33 GC

Libraries were constructed as described in Figure 3 PCR-free libraries from 100 ng and amplified libraries from 1ng of the AMC Low pass

sequencing was carried out on an Illumina MiSeq For each condition two libraries were constructed one in the 96 AFA-TUBE TPX Plate

and one with microTUBE for DNA Shearing and subsequent transfer to PCR plate for library construction (control library)

Covaris Application Note | wwwcovariscom

6

Whole Genome Sequencing on HiSeq X TenPCR-free WGS libraries were constructed as described in Figure 3 using 200 ng Coriell NA12878 genomic DNA For each condition two

libraries were constructed one entirely in the 96 AFA-TUBE TPX Plate and one with microTUBE for DNA Shearing and subsequent transfer

to PCR plate for library construction (control library) Sequencing was carried out by Novogene on the Illumina HiSeq X Ten platform Post

sequencing following exclusion filters were applied before analysis ( exclusion for AFA-TUBE and microTUBE libraries respectively)

- Low mapping quality reads 47 and 47

- Duplicate exclusion 83 and 79

- Low base quality 45 and 42

- Second observation from an insert with overlapping reads 2 and 41

Table 2 WGS PCR-free Libraries

Input Amplification DNA Covaris Vessel Mean Fragment size (target) Number of replicates

200 ng PCR-free NA1287896 AFA-TUBE TPX Plate

350 bp 4microTUBE

Alignment Summary Table

WGS Coverage Table

Table 3 WGS coverage and alignment tables

Mean Coverage

MedianCoverage PCT_5X PCT_10X PCT_15X PCT_20X PCT_25X

96 AFA-TUBE TPX Plate

341 34 981 978 974 965 918

microTUBE 326 33 980 977 973 960 892

Total Reads PF_READS_ALIGNED

PCT_PF_READS _ALIGNED

PF_HQ_ALIGNED _BASES

PF_HQ_ALIGNED _Q20_BASES

PCT_READS_ALIGNED _IN_PAIRS

PCT_ADAPTER_ Dimer

96 AFA-TUBETPX Plate

855785748 854357548 9983 60306787476 58766369045 9886 001

microTUBE 833328788 831857134 9982 58623160032 57180420381 9903 001

Covaris Application Note | wwwcovariscom

7

96 AFA-TUBE TPX Plate

96 AFA-TUBE TPX Plate

microTUBE

microTUBE

Figure 6 Picard plots of 200 ng PCR-free WGS libraries

0 20 40 60 80 100

00

05

10

15

20

All Reads Level All Reads GC Bias Plot

GC of 100 base windows

Frac

tion

of n

orm

aliz

ed c

over

age

010

2030

40

Mea

n ba

se q

ualit

y

minus

Normalized CoverageWindows at GCBase Quality at GC

0 20 40 60 80 100

00

05

10

15

20

All Reads Level All Reads GC Bias Plot

GC of 100 base windows

Frac

tion

of n

orm

aliz

ed c

over

age

010

2030

40

Mea

n ba

se q

ualit

y

minus

Normalized CoverageWindows at GCBase Quality at GC

Figure 7 Cumulative Probability Distributions of Avg Depth of Coverage of the Bad Promoters

Figure 8 Relative ldquoBad Promotersrdquo coverageCharacterizing and measuring bias in sequence data Ross et al Genome Biology 2013 14R51

0102030405060708090

100

Dept

h of

Seq

uenc

ing

Cove

rage

Bad Promoters

Average Coverage oneTUBE Average Coverage microTUBE

Figure 9

AFA-TUBE

Figure 8Bad Promoters List Analysis for WGS DataldquoCharacterizing and measuring bias in sequence datardquo from Ross et al describes a set of 1000 promoters with the lowest relative coverage

(based on an Illumina data set) and called this list the lsquobad promotersrsquo list These ldquoBad Promoters (BP)rdquo are GC-rich (averaging 79 GC

composition)

Covaris Application Note | wwwcovariscom

8

USA Covaris Inc | Tel +1 7819323959 | Fax +1 7819328705 | Email customerservicecovariscomEurope Covaris Ltd | Tel +44 (0)845 872 0100 | Fax +44 (0)845 384 9160 | Email emeacustomerservicecovariscomWeb wwwcovariscom | Applications applicationsupportcovariscom | Service and Support techsupportcovariscomM020072_RevB_Apr2019 | Information subject to change without notice | For research only | Not for use in diagnostic procedures | 2019copy Covaris Inc

Stay Connected

ConclusionCombination of DNA shearing in the AFA-TUBE with Swift Biosciences Accel-NGS 2S Kit enables for the first time DNA shearing end repair

and adapter ligation in the same vessel thereby eliminating cumbersome sample transfers and enabling a streamlined workflow Sequencing

performance metrics in AFA-TUBE are matching or exceeding current benchmark consisting of shearing in microTUBE and then transferring into a

PCR plate for library construction

DNA Shearing with the 96 AFA-TUBE TPX Plate demonstrates robust performances For both conditions analyzed 150 and 350 bp the 48

technical replicates display highly reproducible and tight DNA fragment size distributions Testing performances with different DNA mass also

highlight that as expected with mechanical DNA shearing the protocols are robust across a wide input range

Artificial Microbial Community (AMC) Sequencing data show no adverse biases in coverage uniformity with PCR-free and PCR libraries at inputs

ranging from 1 ng to 100 ng and DNA from species with varying GC

Whole Genome Sequencing on HiSeq X Ten shows an even coverage across the human genome and similar insert size distribution between

AFA-TUBE and microTUBE libraries ldquoBad Promotersrdquo list in depth analysis shows an equivalent coverage between microTUBE and AFA-TUBE

Visit wwwcovariscom and wwwswiftbioscicom for more information

Covaris Application Note | wwwcovariscom

6

Whole Genome Sequencing on HiSeq X TenPCR-free WGS libraries were constructed as described in Figure 3 using 200 ng Coriell NA12878 genomic DNA For each condition two

libraries were constructed one entirely in the 96 AFA-TUBE TPX Plate and one with microTUBE for DNA Shearing and subsequent transfer

to PCR plate for library construction (control library) Sequencing was carried out by Novogene on the Illumina HiSeq X Ten platform Post

sequencing following exclusion filters were applied before analysis ( exclusion for AFA-TUBE and microTUBE libraries respectively)

- Low mapping quality reads 47 and 47

- Duplicate exclusion 83 and 79

- Low base quality 45 and 42

- Second observation from an insert with overlapping reads 2 and 41

Table 2 WGS PCR-free Libraries

Input Amplification DNA Covaris Vessel Mean Fragment size (target) Number of replicates

200 ng PCR-free NA1287896 AFA-TUBE TPX Plate

350 bp 4microTUBE

Alignment Summary Table

WGS Coverage Table

Table 3 WGS coverage and alignment tables

Mean Coverage

MedianCoverage PCT_5X PCT_10X PCT_15X PCT_20X PCT_25X

96 AFA-TUBE TPX Plate

341 34 981 978 974 965 918

microTUBE 326 33 980 977 973 960 892

Total Reads PF_READS_ALIGNED

PCT_PF_READS _ALIGNED

PF_HQ_ALIGNED _BASES

PF_HQ_ALIGNED _Q20_BASES

PCT_READS_ALIGNED _IN_PAIRS

PCT_ADAPTER_ Dimer

96 AFA-TUBETPX Plate

855785748 854357548 9983 60306787476 58766369045 9886 001

microTUBE 833328788 831857134 9982 58623160032 57180420381 9903 001

Covaris Application Note | wwwcovariscom

7

96 AFA-TUBE TPX Plate

96 AFA-TUBE TPX Plate

microTUBE

microTUBE

Figure 6 Picard plots of 200 ng PCR-free WGS libraries

0 20 40 60 80 100

00

05

10

15

20

All Reads Level All Reads GC Bias Plot

GC of 100 base windows

Frac

tion

of n

orm

aliz

ed c

over

age

010

2030

40

Mea

n ba

se q

ualit

y

minus

Normalized CoverageWindows at GCBase Quality at GC

0 20 40 60 80 100

00

05

10

15

20

All Reads Level All Reads GC Bias Plot

GC of 100 base windows

Frac

tion

of n

orm

aliz

ed c

over

age

010

2030

40

Mea

n ba

se q

ualit

y

minus

Normalized CoverageWindows at GCBase Quality at GC

Figure 7 Cumulative Probability Distributions of Avg Depth of Coverage of the Bad Promoters

Figure 8 Relative ldquoBad Promotersrdquo coverageCharacterizing and measuring bias in sequence data Ross et al Genome Biology 2013 14R51

0102030405060708090

100

Dept

h of

Seq

uenc

ing

Cove

rage

Bad Promoters

Average Coverage oneTUBE Average Coverage microTUBE

Figure 9

AFA-TUBE

Figure 8Bad Promoters List Analysis for WGS DataldquoCharacterizing and measuring bias in sequence datardquo from Ross et al describes a set of 1000 promoters with the lowest relative coverage

(based on an Illumina data set) and called this list the lsquobad promotersrsquo list These ldquoBad Promoters (BP)rdquo are GC-rich (averaging 79 GC

composition)

Covaris Application Note | wwwcovariscom

8

USA Covaris Inc | Tel +1 7819323959 | Fax +1 7819328705 | Email customerservicecovariscomEurope Covaris Ltd | Tel +44 (0)845 872 0100 | Fax +44 (0)845 384 9160 | Email emeacustomerservicecovariscomWeb wwwcovariscom | Applications applicationsupportcovariscom | Service and Support techsupportcovariscomM020072_RevB_Apr2019 | Information subject to change without notice | For research only | Not for use in diagnostic procedures | 2019copy Covaris Inc

Stay Connected

ConclusionCombination of DNA shearing in the AFA-TUBE with Swift Biosciences Accel-NGS 2S Kit enables for the first time DNA shearing end repair

and adapter ligation in the same vessel thereby eliminating cumbersome sample transfers and enabling a streamlined workflow Sequencing

performance metrics in AFA-TUBE are matching or exceeding current benchmark consisting of shearing in microTUBE and then transferring into a

PCR plate for library construction

DNA Shearing with the 96 AFA-TUBE TPX Plate demonstrates robust performances For both conditions analyzed 150 and 350 bp the 48

technical replicates display highly reproducible and tight DNA fragment size distributions Testing performances with different DNA mass also

highlight that as expected with mechanical DNA shearing the protocols are robust across a wide input range

Artificial Microbial Community (AMC) Sequencing data show no adverse biases in coverage uniformity with PCR-free and PCR libraries at inputs

ranging from 1 ng to 100 ng and DNA from species with varying GC

Whole Genome Sequencing on HiSeq X Ten shows an even coverage across the human genome and similar insert size distribution between

AFA-TUBE and microTUBE libraries ldquoBad Promotersrdquo list in depth analysis shows an equivalent coverage between microTUBE and AFA-TUBE

Visit wwwcovariscom and wwwswiftbioscicom for more information

Covaris Application Note | wwwcovariscom

7

96 AFA-TUBE TPX Plate

96 AFA-TUBE TPX Plate

microTUBE

microTUBE

Figure 6 Picard plots of 200 ng PCR-free WGS libraries

0 20 40 60 80 100

00

05

10

15

20

All Reads Level All Reads GC Bias Plot

GC of 100 base windows

Frac

tion

of n

orm

aliz

ed c

over

age

010

2030

40

Mea

n ba

se q

ualit

y

minus

Normalized CoverageWindows at GCBase Quality at GC

0 20 40 60 80 100

00

05

10

15

20

All Reads Level All Reads GC Bias Plot

GC of 100 base windows

Frac

tion

of n

orm

aliz

ed c

over

age

010

2030

40

Mea

n ba

se q

ualit

y

minus

Normalized CoverageWindows at GCBase Quality at GC

Figure 7 Cumulative Probability Distributions of Avg Depth of Coverage of the Bad Promoters

Figure 8 Relative ldquoBad Promotersrdquo coverageCharacterizing and measuring bias in sequence data Ross et al Genome Biology 2013 14R51

0102030405060708090

100

Dept

h of

Seq

uenc

ing

Cove

rage

Bad Promoters

Average Coverage oneTUBE Average Coverage microTUBE

Figure 9

AFA-TUBE

Figure 8Bad Promoters List Analysis for WGS DataldquoCharacterizing and measuring bias in sequence datardquo from Ross et al describes a set of 1000 promoters with the lowest relative coverage

(based on an Illumina data set) and called this list the lsquobad promotersrsquo list These ldquoBad Promoters (BP)rdquo are GC-rich (averaging 79 GC

composition)

Covaris Application Note | wwwcovariscom

8

USA Covaris Inc | Tel +1 7819323959 | Fax +1 7819328705 | Email customerservicecovariscomEurope Covaris Ltd | Tel +44 (0)845 872 0100 | Fax +44 (0)845 384 9160 | Email emeacustomerservicecovariscomWeb wwwcovariscom | Applications applicationsupportcovariscom | Service and Support techsupportcovariscomM020072_RevB_Apr2019 | Information subject to change without notice | For research only | Not for use in diagnostic procedures | 2019copy Covaris Inc

Stay Connected

ConclusionCombination of DNA shearing in the AFA-TUBE with Swift Biosciences Accel-NGS 2S Kit enables for the first time DNA shearing end repair

and adapter ligation in the same vessel thereby eliminating cumbersome sample transfers and enabling a streamlined workflow Sequencing

performance metrics in AFA-TUBE are matching or exceeding current benchmark consisting of shearing in microTUBE and then transferring into a

PCR plate for library construction

DNA Shearing with the 96 AFA-TUBE TPX Plate demonstrates robust performances For both conditions analyzed 150 and 350 bp the 48

technical replicates display highly reproducible and tight DNA fragment size distributions Testing performances with different DNA mass also

highlight that as expected with mechanical DNA shearing the protocols are robust across a wide input range

Artificial Microbial Community (AMC) Sequencing data show no adverse biases in coverage uniformity with PCR-free and PCR libraries at inputs

ranging from 1 ng to 100 ng and DNA from species with varying GC

Whole Genome Sequencing on HiSeq X Ten shows an even coverage across the human genome and similar insert size distribution between

AFA-TUBE and microTUBE libraries ldquoBad Promotersrdquo list in depth analysis shows an equivalent coverage between microTUBE and AFA-TUBE

Visit wwwcovariscom and wwwswiftbioscicom for more information

Covaris Application Note | wwwcovariscom

8

USA Covaris Inc | Tel +1 7819323959 | Fax +1 7819328705 | Email customerservicecovariscomEurope Covaris Ltd | Tel +44 (0)845 872 0100 | Fax +44 (0)845 384 9160 | Email emeacustomerservicecovariscomWeb wwwcovariscom | Applications applicationsupportcovariscom | Service and Support techsupportcovariscomM020072_RevB_Apr2019 | Information subject to change without notice | For research only | Not for use in diagnostic procedures | 2019copy Covaris Inc

Stay Connected

ConclusionCombination of DNA shearing in the AFA-TUBE with Swift Biosciences Accel-NGS 2S Kit enables for the first time DNA shearing end repair

and adapter ligation in the same vessel thereby eliminating cumbersome sample transfers and enabling a streamlined workflow Sequencing

performance metrics in AFA-TUBE are matching or exceeding current benchmark consisting of shearing in microTUBE and then transferring into a

PCR plate for library construction

DNA Shearing with the 96 AFA-TUBE TPX Plate demonstrates robust performances For both conditions analyzed 150 and 350 bp the 48

technical replicates display highly reproducible and tight DNA fragment size distributions Testing performances with different DNA mass also

highlight that as expected with mechanical DNA shearing the protocols are robust across a wide input range

Artificial Microbial Community (AMC) Sequencing data show no adverse biases in coverage uniformity with PCR-free and PCR libraries at inputs

ranging from 1 ng to 100 ng and DNA from species with varying GC

Whole Genome Sequencing on HiSeq X Ten shows an even coverage across the human genome and similar insert size distribution between

AFA-TUBE and microTUBE libraries ldquoBad Promotersrdquo list in depth analysis shows an equivalent coverage between microTUBE and AFA-TUBE

Visit wwwcovariscom and wwwswiftbioscicom for more information