SWIFT AMPLICON® SARS-CoV-2 RESEARCH PANELProtocol for Cat. No.: AL-COV48

isit swift iosci com protocols for updates

Tec otes: • Primerclip: A Tool for r Primer Sequences Using Command Line or

Galaxy

www.swiftbiosci.com Version 2.0

Version 200506

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About This GuideThis guide provides instructions for the preparation of targeted NGS li raries from DNA samples using an Accel-Amplicon panel

P N a t t r Protocol for s s c all t s ct o s rta to Pro ct

for at o t Co t ts at r als a t Not cl a t at r al Co s rat o s.

Product InformationThis panel ena les the preparation of high uality targeted ne t-generation se uencing (NGS) li raries from a variety of sample types Adapters are available for dual inde ing and multiple ing up to samples on a se uencing run The single-tu e wor flow from cDNA to li rary can e completed within two hours

The it provides the primer pool, li rary preparation reagents, and Illumina compati le adapters and has een validated on Illumina platforms The ta le on the ne t page lists ey characteristics, RT recommendations, cycling conditions, and typical performance of this

panel

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* or multiple ing over -ple please refer to our wift Amplicon - or flow

https://swiftbiosci.com/wp-content/uploads/2019/05/384-pr-4.pdf

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Accel-Amplicon Panel Work lows rotocol co ta s a lt l PC st for t s lta o s ro ct o of r s of

a l co tar ts a s l t a a st for t a t o of al a a t rs a l lt l of to -384 l rar s.

a as cl a s ar s to r f t sa l r o s ol o cl ot s a c a ff r co os t o t st s.

DNA

Dual-Indexed Amplicon Library

Indexed Sequencing Adapters

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Kit Contentscc l l co a ls ar a a la l a ac a s t r a ts c ss ol for

t r arat o of l rar s.

Kit ReagentsQuantity (μl)

Storage (ºC)48 rxn

Multiplex PCR(Pre-PCR)

a t

a t

Pr PC

Indexing Step(Post-PCR)

ac of

ac of

ff r

Post PC

* eagent is the panel-specific set of multiple amplification primers

Kit Reagent Quantity (μl) 48 rxns Storage (ºC)

Additional Components Included

P NaCl ol t o Room Temp

P N Plac t s o c for at l ast t s to allo s to r ac C r or to tt .

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Materials and Equipment Not Included• a t c a s for cl a st s . . P s l ct a s c a Co lt r Cat. No.

or P r P a s c a Co lt r Cat. No.

• a t c rac for cl a st s . . tro a a or co rt P Plat• rar a t f cat o t PC as• t or ot r fl oro tr c as assa s for t r N co c trat o• . PC t s str s or ll lat s• C tr f co at l t for at of last c co s a l s• Pro ra a l t r oc cl r• rosol r s sta t lo r t t o tt s a t s to • roof a sol t t a ol ol c lar olo ra• N cl as fr at r ol c lar olo ra

Storage and Usage Warning

pon receipt, store the Accel-Amplicon panel it at -20 C with the e ception of PEG solution, which should e stored at room temperature

Separate the Multiple PCR Reagents ( eep in pre-PCR area) and Inde ing Reagents ( eep in post-PCR area)

To ma imi e use of en yme when ready to use, remove en yme tu es from -20 C storage and place on ice for at least 10 minutes to allow en ymes to reach 4 C prior to pipetting

Plan to prepare a minimum of 6 reactions for a 24-reaction it or 24 reactions for a 6-reaction it to avoid e cessive reagent loss from preparing 4 master mi es with 5-10 overage each

After thawing reagents to 4 C, riefly vorte (e cept the en ymes) to mi them well En yme G3 is the only en yme that may e gently vorte ed Spin all tu es in a microfuge to collect contents prior to opening

Always add reagents to the master mi in the specified order as stated throughout the Protocol The dual inde ed adapters are the only reagents that are added individually to each sample

IMPORTANT! ss l all r a t ast r s a r act o s N C a scal ol s as

a ro r at s c ss ol to co sat for tt loss. o calc lat t total ol of t ast r s s o r cc l l co ast r ol Calc lator a r ar t a a c to s r t a t c a s o ot o r r

r s s l ct o st s l a a t co l t o of ast r ass l . N l ct to stor ast r s a r a ts o c r or to c at o s r c s

l s a rfor a c of t s ro ct.

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Tips and Techniques

• P s call s arat t la orator s ac t a s l s r r PC a ost PC roc ss s ar rfor cl a ro r at r a t o s for lt l PC a

t .• Cl a la ar as s . so oc lor t l ac .• s arr r tt t s to a o os r to ot t al co ta a ts.• l a s c a t s t ac sa l .• o sa l s to ost PC ar a for o t s.• r ar N C co trols l as s s a s al N C s for r N sa l s

to t oo .

cc l l co l a a l co r c t t c olo os s a r s of co ta at o of s rfac s a ot r sa l s follo t a l f cat o st . Pl as s tr ca t o o o r sa l t s follo t lt l PC st . t s l r co t at s arat or s ac s a tt s a ta for r PC a ost PC st s. at

r ss r oo s o l s for ost PC st s f a a la l . Cl a la ar as s . so oc lor t l ac a s s c alt arr r tt t s. s os of tt t s a ot r s osa l s s al last c a s.

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compensate for pipetting loss. To calculate the total volume of the master mixes based on the number of reactions of choice, use our

. This took automatically incorporates 5% excess volume to compensate for pipetting loss.

To assemble reagent master mixes for the Multiplex PCR and Indexing steps, ensure the a nt ia a t a an t n to on ic A t t a in a nt i o t c pt the enzymes) to mix them well. Enzyme G3 s ould e gently armed to room

temperature and nu a ed for minu es in order to dissolve e solutes. Spin tubes in a microfuge to collect contents prior to opening. Add reagents in the order listed when preparing master mix. Master mixes should be prepared and stored ON ICE until used.

IMPORTANT! Prepare the reagents in advance to ensure the magnetic beads do not dry out during size selection steps. Always add reagents in specified order. This applies to all reagents except for the indexed adapters that should be added individually to uniquely inde each library.

Ensure PEG NaCl solution is at room temperature.

3. pa a t ano o ution u in p oo a o ut t ano an nuc a at App o i at o t ano o ution i u p a p

Prepare the Reagent Master Mixes and Ethanol o c at a a t i ca a nt o u a app op iat u in c o u to

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BEGIN YOUR ACCEL-AMPLICON PROTOCOL

Prepare the DNA Libraries

Pre-Program Thermocycler

ta o to p p o a t t oc c o t u tip Not pan p ci c c c an In in t p NOTE: Cycling conditions and data ua it can a a

on input ua it an uantit

IMPORTANT! Work in pre-PCR area.

Multiplex PCRThermocycler Program

IndexingThermocycler Program

Lid heating OFF

in

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Multiplex PCR StepLoad the Multiplex PCR program and allow the block to reach 98 °C before loading samples con i i atin i tu n N an i t to ac

oa o a p DNA a u t it into ac tu p on ic

Keep all tubes on ice during assembly of the master-mix and the reaction until placed in thermocycler. Make the Multiplex PCR Reaction Mix. Components G1and G2 should

o t i t efore adding o e mas er mix n yme G s ould e gen ly thawed o room empera ure and nu a ed for minu es in order o dissol e all solu es efore adding o e mas er mix Place on ice for remainder of use.

Panel-Specific Multiplex PCR Reaction MixTo calculate the total volume of the master mixes based on the number of reactions of choice, use our Accel-Amplicon Master Mixing Volume Calculator.

Component Volume (1 Reaction)

Reagent G1*

Reagent G2

Enzyme G3

Reaction Mix 20 μl

*Reagent G1 is the panel-specific set of amplification primers.

4. i t a t i an t n a o t u tip action i to ac input DNA sample. Place in a pre ea ed thermocycler and run the program.

IMPORTANT! Move samples to post-PCR area before opening tubes. Keep samples at room tempera-ture. At no time should ‘with bead’ samples be stored on ice, as this affects binding to magnetic beads.

5. Near the completion of the thermocycler run, prepare the Indexing Reaction Mix in the post-PCR area with the following components. Assemble this reaction mix on ice and keep cold until adding it to samples in the Indexing Step. All components may be master-mixed when running multiple samples in parallel.

Indexing Step To calculate the total volume of the master mixes based on the number of reactions of choice, use our Accel-Amplicon Master Mixing Volume Calculator.

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Component Volume (1 Reaction)

Buffer Y1

Enzyme Y2

Enzyme Y3

Enzyme Y4

Reaction Mix 35 μl

Size Selection and Clean-Up Step 1n u a an a p a at oo t p atu i o t a to o o ni

before use.

A atio o a n tic a to ac a p i o t in u pin the samples in a microfuge. Ensure no bead-sample suspension droplets are left on the sides of the tube.

Incubate the samples for 5 minutes at room temperature off the magnet.

ac t a p tu on a a n tic ac unti t o ution c a an a p t i o 5 minutes).

While leaving your sample on the magnet, remove and discard the supernatant without dis-turbing the pellet (approximately 5 µl may be left behind). Leave tubes on the magnet.

A o p pa t ano o ution to t p t i it i ti on t a n t ca not to i tu t p t Incu at o con an t n ca u o t

ethanol solution.

Repeat step 11 once for a second wash with the ethanol solution.

Pulse-spin the samples in a microfuge, place back onto the magnet and remove any residual ethanol solution from the bottom of the tube with a small volume tip. Proceed to the Indexing Step.

IMPORTANT! Continue working in the post-PCR area.

Indexing StepLoad the Indexing Thermocycler Program and allow the block to reach 37 °C before loading samples.

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(

Place in the thermocycler at °C for for t s with li eating or t t r oc cl r l o .

Size Selection and Clean-Up Step 2n u Na o ution i at oo t p atu i o t t Na o ution to

homogenize before use.

A atio o Na o ution to ac a p i o t in n u no bead-sample suspension droplets are left on the sides of the tube.

Incubate the samples for 5 minutes at room temperature off the magnet.

Pulse-spin the samples in a microfuge. Place the sample tubes on a magnetic rack until the o ution c a an a p t i o inut

While leaving your sample on the magnet, remove and discard the supernatant without dis-turbing the pellet (approximately 5 µl may be left behind). Leave tubes on the magnet.

A o p pa t ano o ution to t p t i it i ti on t a n t ca not to i tu t p t Incu at o con an t n ca u o t

t ano solution.

Repeat step 23 once for a second wash with the ethanol solution.

Pulse-spin the samples in a microfuge, place back onto the magnet and remove any residual ethanol solution from the bottom of the tube with a small volume tip.

at l o o t u an u p n t p t i in well by pipetting up and down until homogenous. Incubate at room temperature for 2 minut o t a n t n p ac t a p ac on t a n t for approxima ely minu es or un il e solu ion clears and and a pelle is formed

ra sf r t cl a l l rar l at to a fr s t . s r t l at o s ot co ta a t c a s cat ro coloration). f a t c a s ar r s t lac o a t a tra sf r l at to a fr s t .

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i rary Quantificationrar a t f cat o PC s r r for acc rat a t f cat o of l rar s. uantify a

1 10,000 dilution of the li rary in triplicate using a PCR- ased assay for st r s lts. co ct o t t PC Ct al s l as s a li rary si e of 265 to calc lat l rar

olar t e sure to ad ust for proper li rary si e of the standards in your li rary uantification it for acc rat r s lts.

Pl as ot :

Sequence the DNA LibrariesPlease refer to the latest version of Illumina E periment Manager for detailed instructions on how to set up a sample sheet Be sure to select the appropriate wor flow parameters as noted elow

• Read Type: “Paired End” • Cycle Read 1 151 , Cycles Read 2 151

• ca s there is no PCR enrichment of the li rary following the Inde ing Step, electrophoretic or fl oro t c a t f cat o methods ca ot st s f ll rs s art all a a t l rar a r s lts ll acc rat .

• a t o t f al l rar str ct r s art all s l stra c slo s rat o l a to acc rat s t r at o a a t f cat o ct o .

ake sure t e se apter Trimming an se apter Trimming ea are selecte Please ensure that adapter trimming is ena led while setting up the se uencing run Failure to trim adapter se uences will result in incorrect primer trimming and will lead to inaccurate variant calling To overcome this issue, ena le automatic trimming y the se uencer software or perform adapter trimming y Trimmomatic during data analysis For more information, please consult our Bioinformatics Resources page at swift iosci com iof

PhiX Spike-In:

ll cc l l co catalo a ls a al at t o t P o ot a . or sequencing on the N t l as co s lt ll a s r co at o s for P

s s s c lo rs t a l co l rar s.

swift%F4%80%80%82iosci%F4%80%80%83com%F4%80%80%84%F4%80%80%82iof%F4%80%80%86

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Adapter and Primer Trimming

Please ensure that adapter trimming is ena led while setting up the se uencing run Alternatively, adapter trimming ca e performed ioinformatically prior to analysis In addition, Accel-Amplicon are designed with overlapping amplicons to allow for contiguous regions of coverage in a single-tu e format Therefore, synthetic primer se uences will e encountered oth at the eginning and end of some reads, which must e trimmed during the first step in data analysis This can e done using a pu licly availa le tool called Primerclip (http githu com swift iosciences primerclip)

For information r ar ar a t call tools and for panel-specific files, please consult our Bioinformatics Resources page at swift iosci com iof

http://github.com/swiftbiosciences/primerclipswiftbiosci.com/biofx

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Appendix

Section A: Library Multiplexing Options for MiSeqSwift Recommends an average sequencing depth of 5000X for somatic variant detecting down to 1% allele frequency and 200-500X for germline variant detection. Use the following equation to determine possible number of libraries to multiplex per sequencing run:

Level of multiplexing = (number of paired-end reads) / (number of amplicons * intended average read depth)

*For multiplexing over 96-plex, please refer to our Swift Amplicon 384-PLEX Workflow.

ec i n B a e ia a e a a heePlease refer to Accel-Amplicon products material safety data sheet (MSDS) for more information a out the potential ha ards for each component (reagents, uffers and en ymes) and instruc-tions on safe use

ection C: Notes for AutomationThis protocol is readily automata le A 10 overage volume of reagents is supplied to accommodate automation Please contact us at automation swift iosci com if you re uire additional reagent overage volume or would li e to learn a out our custom pac aging options

hile Swift Biosciences does not supply automated li uid handling instruments or consuma les, our automation team colla orates with automation solution providers and customers to develop and ualify optimi ed automated scripts for use of our its with li uid handling platforms routinely used in NGS li rary preparation Please contact us at automation swift iosci com to discuss automating your Accel-Amplicon Panel with your particular automated li uid handling system

https://swiftbiosci.com/quality-information/

Section D: Indexed Adapter SequencesDuring the Inde ing Step in the protocol, you must use a uni ue com ination of Inde Adapters to re-suspend and la el each li rary Li raries made with uni uely inde ed adapter com inations may e multiple ed during cluster generation and co-se uenced on the same Illumina flow cell

C T TS: ni ue inde ed adapters, which should e used where this manual calls for 5 or 10 l of each Inde Primer

D5 Adapters Sequence MiSeq, HiSeq 2000/2500Sequence MiniSeq, NextSeq,

HiSeq 3000/4000

D TATAGCCT AGGCTATA

D ATAGAGGC GCCTCTAT

D CCTATCCT AGGATAGG

D GGCTCTGA TCAGAGCC

D AGGCGAAG CTTCGCCT

D TAATCTTA TAAGATTA

D CAGGACGT ACGTCCTG

D GTACTGAC GTCAGTAC

T : Include reverse compliment se uences provided in the ta le a ove when using Illumina MiniSe , Ne tSe , or iSe 3000 4000 systems

D7 Adapters Sequence

D ATTACTCG

D TCCGGAGA

D CGCTCATT

D GAGATTCC

D ATTCAGAA

D GAATTCGT

D CTGAAGCT

D TAATGCGC

D CGGCTATG

D TCCGCGAA

D711 TCTCGCGC

D712 AGCGATAG

The num er on the product tu e la el indicates which inde ed adapter is provided in the tu e During li rary prep, ma e sure to note which inde ed adapter com ination you are using with your sample and do not use the same inde ed adapter com ination on two different samples you plan to co-se uence

For greater than 6-ple multiple ing, please refer to our Swift Amplicon 384-PLE or flow

15

https://swiftbiosci.com/wp-content/uploads/2019/11/PRT-004-384-Protocol-Addendum-Rev-1.pdf

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Section E: Helpful Information and Troubleshooting

Problem Possible Cause Suggested Remedy

Lower than expected yields

Inadequate sample quality and/or quantity, incorrect input quan-ti cation t o o inco ct SPRI™ methods.

more input an extend the incubation time for the Index-in t p

o inut to inut Perform SPRI carefully.

Unusual electropho-retic trace

Secondary structure of adapters and lack of PCR enrichment of the library following the Indexing Step exhibit characteristics of migration artifacts.

Quantify library with a qPCR-based method; if you need to ascertain amplicon insert size from the sequencing data. (Review full explanation in “Structure of Amplicon Libraries and Migration Behavior” section.)

Precipitates in En-zyme G3

Salt precipitation.Allow the vial to reach room temperature and gently nutate to dissolve solids. Place on ice for remainder of use.

Lower than expected cluster density

o in i a uanti cation Bioanalyzer and Qubit do not accurately quantify fully adapted library vs. other DNA.

Quantify library with a qPCR-based method o o c oa in ca cu ation

Incomplete resuspen-sion of beads after ethanol wash during SPRI™ steps

Over-drying of beads.Continue pipetting the liquid over the beads to break up clumps for complete resuspension.

Shortage of enzyme reagents

ip ttin n at in t a o

A o n a nt to ui i at to o inut p io to pip ttin

Retention of liquid in pipette tip

Viscous reagents may stick to pipette tip, especially for non-low retention tips.

Pipette up and down several times to ensure all liquid and/or beads are released from the pipette tip.

If you experience problems with your library prep, please contact us at TechnicalSupport@swiftbio.com, or p on at a p

on a i a

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“A+B” is an extended amplicon, which is a contiguous alignable sequence

Section F: Structure of Accel-Amplicon Libraries and Migration Behavior The secondary structure of Accel-Amplicon li raries e hi its two features, which should e understood if analy ed using electrophoretic methods such as Agilent Bioanaly er or TapeStation

. If using high ol c lar t DNA, e tended amplicons can e o served They are formed from the forward primer and the reverse primer of two ad acent amplicons Note that these e tended amplicons are not formed when using fragmented or cross-lin ed (FFPE) DNA, or cell-free DNA Coverage uniformity is not affected y the presence or a sence of e tended amplicons

A B

A+B

. After inde ing, the li rary is partially single-stranded and the migration is impaired, causing the li rary to appear large on the Bioanaly er therefore, the traces should not e used to accurately determine the si e or the uantity of the li rary

Bioanalyzer(duplicate)

Bioanalyzer(duplicate)

DNA

Dual-Indexed Amplicon Library

Indexed Sequencing Adapters

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General Warrantyi t io ci nc Inc i t a ant t at it p o uct t i t p ci cation at t ti o i Any

sample or model used in connection with Swift’s product literature is for illustrative purposes only and does not constitute a warranty that the products will conform to the sample or model.

To the maximum extent permitted by applicable law, Swift hereby expressly disclaims, and the buyer hereby expressly waives, any warranty regarding results obtained through the use of the products including, without limitation, any claim of inaccurate, invalid, or incomplete results. All other warranties, representations, terms and conditions (statutory, express, i p i o ot i a to ua it con ition c iption c anta i it tn o pu po o non in in nt c pt for the implied warranty of title) are hereby expressly excluded.

A a ant c ai on p o uct u t a in itin it in nin t a o c ipt o t p o uct i t o liability and the buyer’s exclusive remedy for a breach of this warranty is limited to replacement or refund at the sole option of Swift.

The warranties identified in this paragraph are Swift’s sole and exclusive warranties with respect to the products and are in lieu of all other warranties, statutory, express or implied, all of which other warranties are expressly disclaimed, including without limitation any implied warranty of merchantability, fitness for a particular purpose, non-infringement, or regarding results obtained through the use of any product (including, without limitation, any claim of inaccurate, invalid or incomplete results), whether arising from a statute or otherwise in law or from a course of performance, dealing or usage of trade.

Limitation of LiabilitySwift Biosciences, Inc. (“Swift”) shall have no liability under the warranties cited above with respect to any defect in the p o uct a i in o i p ci cation o at ia upp i t u ii i u a a o n i nc o t u o its employees or agents; (iii) abnormal working conditions at the buyer’s premises; (iv) failure to follow Swift’s use restric-tions or instructions (whether oral or in writing); (v) misuse or alteration of the products without Swift’s approval; or (vi) if the buyer is in breach of its payment obligations in regards to purchasing the products.

To the fullest extent allowed by law, in no event shall Swift be liable, whether in contract, tort, strict liability, negligence, warranty, or under any statute or on any other basis for any special, incidental, indirect, exemplary, punitive, multiple or consequential damages sustained by the buyer or any other person or entity arising out of or caused by product, Swift’s performance or failure to perform its obligations relating to the purchase of product or performance of services, Swift’s breach of these terms, the possession or use of any product, or the performance by Swift of any services, whether or not foreseeable and whether or not Swift is advised of the possibility of such damages, including without limitation damages arising from or related to loss of use, loss of data, downtime, procurement of substitute products or services, or for loss of revenue, profits, goodwill, or business or other financial loss.

The total liability of Swift arising under or in connection with the purchase of the products, including for any breach of contractual obligations and/or any misrepresentation, misstatement or tortious act or omission (including without limita-tion, negligence and liability for infringement of any third party intellectual property rights) shall be limited to damages in an amount equal to the amount paid to Swift under the purchase agreement.

The exclusion of liability shall apply only to the extent not prohibited by applicable law.

Notice to Purchaser: Limited LicenseThis product is for research use only and is licensed to the user under Swift Biosciences intellectual property only for the purchaser’s internal purposes. Not for use in diagnostic procedures.

Swift Biosciences, Inc. 574 S. Wagner Rd, Suite 100, Ann Arbor, MI 48103. 734-330-2568. www.swiftbiosci.com 20 i t io ci nc Inc i t o o an Swift Amplicon a t a a of Swift Biosciences. Accel-

Amplicon Panels are covered by one or more claims of US Patent No(s). 10,316,359. This product is for Research Use Only. Not for use in diagnostic procedures. Illumina, HiSeq, MiniSeq, MiSeq, and NextSeq are registered trademarks of Illumina, Inc. Agencourt and AMPure are registered trademarks and SPRI, SPRIplate, and SPRIselect are trademarks of Beckman Coulter, Inc. Qubit is a registered trademark and DynaMag is a t a a o o i ci nti c Inc i a i a t a a o io a a o ato i Inc i onuc oti

u nc 2020 I u ina Inc A i t PRT-030 Rev 2