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Swift RNA Library Kit

NGS Library Prep for Stranded RNA-Seq

Protocol for Cat. Nos. R1024 and R1096

Compatible with Swift Indexing Primer Kits:

• Single Indexing Primer Kit Set A (12-plex), Swift Cat. No. X6024

• Combinatorial Dual Indexing Primer Kit (96-plex), Swift Cat. No. X8096

• Swift Combinatorial Dual Indexing Primer Kits Set S1-S4 (192- to 768-plex), Swift Cat. Nos. X85192, X86192, X87192, X88192, X89768

• Swift Unique Dual Indexing Primer Kits (24- to 96-plex), Swift Cat. Nos. X9096, X90384, X9096-PLATE, X90384-PLATE, X92304-PLATE

• Normalase Combinatorial Dual Indexing Primer Kit (96-plex), Swift Cat. No. 68096

o Requires Swift NormalaseTM Kit for complete functionality, Cat. No. 66096

Visit swiftbiosci.com/protocols for updates.

Swift RNA Library Kit Protocol 2

Table of Contents

About This Guide ................................................................................................................................. 3

Swift RNA Workflow ............................................................................................................................ 4

Kit Contents ......................................................................................................................................... 5

Materials and Equipment Not Included ............................................................................................. 6

Notes on Starting Input Material ........................................................................................................ 6

Working with RNA ............................................................................................................................... 6

Notes for RNA-Seq Library Preparation ............................................................................................ 7

Prepare the RNA Libraries .................................................................................................................. 7

RNA Fragmentation ........................................................................................................................................... 7

Reverse Transcription ........................................................................................................................................ 9

SPRI Clean-up ................................................................................................................................................... 9

Adaptase .......................................................................................................................................................... 10

Extension ......................................................................................................................................................... 10

SPRI Clean-up ................................................................................................................................................. 11

Ligation ............................................................................................................................................................ 11

SPRI Clean-up ................................................................................................................................................. 11

Indexing PCR ................................................................................................................................................... 11

SPRI Clean-up ................................................................................................................................................. 12

Expected Results ............................................................................................................................... 13

Library Quantification ....................................................................................................................... 13

Sequence the RNA Libraries ............................................................................................................ 13

Appendix ............................................................................................................................................ 14

Section A: SPRIselect Clean-Up Protocol ....................................................................................................... 14

Section B: Kit Compatibility with Upstream Modules ....................................................................................... 14

Section C: Low-quality and FFPE RNA ........................................................................................................... 15

Section D: Protocol Adjustments for Hybridization Capture ............................................................................ 17

Section E: Normalase Instructions .................................................................................................................. 18

Section F: Expected Results for Alternate Insert Sizes ................................................................................... 19

Section G: Data Analysis and Informatics ....................................................................................................... 20

Section H: Notes for Automation ..................................................................................................................... 20

Section I: Troubleshooting Common Problems ............................................................................................... 21

Section J: Indexed Adapter Sequences .......................................................................................................... 22

Revision History ................................................................................................................................ 26

General Warranty ............................................................................................................................... 26

Limitation of Liability ........................................................................................................................ 26

Notice to Purchaser: Limited License ............................................................................................. 26

Swift RNA Library Kit 3

About This Guide Swift RNA for stranded RNA-Seq enables the preparation of high-complexity next generation sequencing (NGS)

libraries. This protocol utilizes Swift AdaptaseÒ technology to produce high-quality RNA-Seq libraries following first-strand cDNA synthesis. This approach preserves strand-specificity in a highly efficient process without conventional second-strand cDNA synthesis. Swift RNA is suitable for the following sample types:

• ribosomal RNA-depleted RNA

• poly(A)-enriched mRNA

• total RNA

• formalin-fixed, paraffin-embedded (FFPE) RNA*

• RNA with low DV200 scores Swift RNA can accommodate an RNA input range of 100 pg to 100 ng directly into the Library Kit. This corresponds

to a range of 10 ng to 1 µg of total RNA input into an appropriate poly(A)-selection or ribosomal RNA (rRNA) depletion module. The Swift RNA Library Kit does not include poly(A)-selection or rRNA depletion modules. If using one of these upstream modules, please follow the general recommendations below and see further details in Appendix Section B. If using a downstream hybridization capture module, please see Appendix D for required protocol adjustments. *For more information, see the Swift Biosciences Application Note titled “The Swift RNA Library Kit Optimizes RNA-Seq Data Quality and Costs for FFPE Samples.”

Module Recommendations

Poly(A) selection Follow manufacturer’s specifications up to the final elution step. Instead of resuspending mRNA capture beads in the manufacturer-specified buffer, resuspend beads in Swift fragmentation master mix and follow the on bead fragmentation protocol (see pages 7-8).

Ribosomal RNA depletion Follow manufacturer’s specifications up to the final elution step. Instead of eluting

in the manufacturer-specified volume, elute in 7 µL of the manufacturer’s buffer for

a final transfer volume of 5 µL. Follow the off bead fragmentation protocol (see pages 7-8).

This kit utilizes IlluminaÒ-compatible adapter sequences and has been validated on Illumina sequencing platforms using poly(A)-enriched Universal Human Reference (UHR) RNA and human brain mRNA.

Feature Swift RNA Specification

Kit Reaction Sizes 24, 96

Input RNA Required 100 pg – 100 ng (mRNA, rRNA-depleted RNA, total RNA)

10 ng – 1 µg (total RNA into an upstream module*)

Time Required ~4.5 hours

Library insert size 200-250, 250-300, 300-350 bp

Indexing options Single, Combinatorial Dual, Unique Dual, Normalase Combinatorial Dual – indexing primer kits and Normalase reagents sold separately

*Note that rRNA depletion and poly(A) enrichment modules are not included with this kit. Swift RNA is compatible with many upstream modules; see Appendix Section B for more details.


IMPORTANT! Read the entire Protocol before use, especially the sections pertaining to Kit Contents,

Materials and Equipment Not Included, Input Material Considerations, and Working with RNA.

Swift RNA Workflow The Swift RNA protocol follows 5 main steps:

1. RNA Fragmentation followed by random priming and reverse transcription generates first-strand cDNA.

2. Swift Adaptase technology simultaneously performs tailing and ligation to incorporate a truncated i7 adapter to the 3’ ends of the cDNA molecules.

3. Extension produces a dsDNA duplex for adapter ligation.

4. Ligation adds a truncated i5 adapter to the 3’ ends of the primer-extended cDNA molecules.

5. Indexing PCR increases library yield, incorporates single or dual indexes, and results in full-length adapters at the ends of each molecule. NOTE: Normalase indexing primers can also be used for compatibility with the Normalase workflow (see Appendix Section E and the Normalase Kit protocol for instructions before starting the Indexing PCR setup).

Bead-based SPRI clean-ups are used throughout to purify the sample by removing unused oligonucleotides and changing buffer composition between steps.

Figure 1: Swift RNA Workflow. RNA-Seq libraries are produced following five main steps: 1) Fragmentation and reverse transcription with a random primer; 2) addition of the i7 adapter using Adaptase; 3) primer extension; 4) addition of the i5 adapter by ligation; and 5) indexing PCR.


Kit Contents The Swift RNA Library Kit contains enough reagents for the preparation of 24 or 96 libraries (10% excess volume provided). Indexing reagents are provided separately in the available Indexing Primer Kits listed below.

Protocol stage Component Volume (µL)

Storage 24 rxns 96 rxns

Fragmentation

• RNA Reagent F1 27 106

-15 °C to -25 °C


• RNA Buffer F3 106 423


Reverse Transcription

• RNA Enzyme R1 27 106

• RNA Enzyme R2 27 106

Adaptase

• RNA Buffer A1 53 212

• RNA Reagent A2 53 212

• RNA Reagent A3 33 132

• RNA Enzyme A4 14 53

• RNA Enzyme A5 14 53

Extension and PCR • RNA Reagent E1 27 106

• PCR Master Mix 1241 4964

Ligation

• RNA Buffer L1 80 317

• RNA Reagent L2 264 1056

• RNA Enzyme L3 53 212

Additional reagents Nuclease-free water 1000 2000

Low EDTA TE 20 mL 20 mL Room Temp

IMPORTANT! Place enzymes on ice for at least 10 min to allow enzymes to reach 4 °C prior to pipetting.

Indexing Reagents Provided Separately (see Appendix Section J for index sequences and volumes)

Reagent Swift Cat. No. Multiplexing Storage

Single Indexing X6024 12

-15 °C to -25 °C

Combinatorial Dual Indexing

X8096 96


X85192, X86192, X87192, X88192, X89768

192-768

Unique Dual Indexing X9096, X90384, X9096-PLATE, X90384-PLATE, X92034-PLATE

24-96

Normalase Combinatorial Dual Indexing

68096 96


Materials and Equipment Not Included

• A compatible Indexing Primer Kit (see above table and Appendix Section J)

• Swift Normalase Kit (Cat. No. 66096), if using the Normalase Indexing Primer Kit (Cat. No. 68096)

• Ribosomal RNA depletion, poly(A)-selection, or hybridization capture module, if required (see Appendix Sections B and D for more information)

• RNaseZap (Invitrogen, Cat. No. AM9780)

• SPRIselect beads (Beckman Coulter, Cat. No. B23317/B23318/B23319) or Agencourt AMPure XP beads (Beckman Coulter, Cat. No. A63880/A63881/A63882)

• Invitrogen DynaMag, Agencourt SPRIPlate or similar magnetic rack for magnetic bead clean-ups

• Qubit, Nanodrop, or similar input RNA quantification assay

• Agilent Bioanalyzer and RNA chip to determine RIN score, if handling low-quality samples

• qPCR-, electrophoretic-, or fluorometric-based library quantification assay for Illumina libraries

• Microcentrifuge

• Programmable thermocycler operating within manufacturer’s specifications

• 0.2 mL PCR tubes or 96-well plate

• Aerosol-resistant tips and pipettes ranging from 1-1000 µL

• 200-proof/absolute ethanol (molecular biology grade) and nuclease-free water for preparation of 80% ethanol

Notes on Starting Input Material

• The Swift RNA Library Kit has been validated at inputs of 100 pg to 100 ng of mRNA and 10 ng to 1 µg of total RNA into an upstream module.

• Please consider transcriptome complexity and sample quality when choosing input RNA quantity. Although libraries may be successfully prepared from low inputs, reduced representation of transcriptome complexity may occur.

• To obtain the best sequence data, make sure that samples are of high quality (RIN ≥ 7) before proceeding. For FFPE or other low-quality RNA (RIN < 7), please see Appendix Section C and contact Swift for further details.

• Since ribosomal RNA (rRNA) makes up ~80-90% of all RNA molecules in a cell, it is recommended to remove rRNA by depletion, perform poly(A) enrichment of mRNA, or enrich for specific target regions using hybridization capture to obtain sufficient RNA-Seq coverage. See Appendix Sections B and D for recommendations on pairing the Swift RNA Library Kit with upstream or downstream modules.

• Ensure RNA samples going directly into the Swift RNA Library Kit are at a volume of 5 µL. See Appendix Section B for recommendations on achieving this volume with compatible upstream modules. If the volume is much

greater than 5 µL, concentrate the sample with a column purification kit, SpeedVac, or other method.

Working with RNA

To reduce the risk of RNA and library contamination, particularly at low inputs:

• DNase treat RNA samples upon isolation.

• Perform all handling of RNA in an RNA-only workstation.

• Clean lab areas and equipment using 0.5% Sodium Hypochlorite (10% bleach) and then treat all surfaces with RNaseZap to reduce the possibility of RNA degradation.

• After coming into contact with surfaces outside of the RNA-only workstation, change gloves or treat gloves with RNaseZap.

• Use barrier pipette tips.

• Physically separate the laboratory space, equipment, and supplies where pre-PCR and post-PCR processes are performed.

To achieve the best results, please follow these guidelines:

• Keep RNA samples on ice and return to -80 °C as soon as possible to reduce sample degradation.

• Minimize the number of freeze-thaw cycles to avoid sample degradation.

• Do not vortex RNA samples. Instead, gently pipet up and down or gently flick to mix.


Notes for RNA-Seq Library Preparation

For best results, please follow these suggestions:

• After thawing reagents, invert or briefly vortex (except enzymes) to mix well. Briefly centrifuge to collect contents.

• If preparing multiple libraries at once, assemble reagent master mixes for each Protocol Stage: Fragmentation, Reverse Transcription, Adaptase, Extension, Ligation, and PCR. Scale master mix volumes as appropriate, using 5-10% excess volume to compensate for pipetting loss. A Master Mix Calculator is available at swiftbiosci.com (.xls format).

• Plan to prepare a minimum of 6 reactions for a 24-reaction kit or 24 reactions for a 96-reaction kit to avoid excessive reagent loss from preparing >4 master mixes with 5-10% overage each.

• Always add enzymes last to master mixes and immediately before adding the master mix to samples.

• Before starting, prepare a fresh 80% ethanol solution for bead-based clean-up steps. Prepare enough for approximately 3 mL per library.

• Preheat each thermocycler program with lid heating ON (~105 °C) prior to the preparation of samples and master mixes.

Prepare the RNA Libraries

NOTE: Please read this protocol before beginning an upstream RNA processing kit (e.g. ribosomal RNA depletion or

poly(A) selection). If using a ribosomal RNA depletion kit, elute the final RNA in a volume of 5 µL for input into the Fragmentation off bead protocol below. If using a poly(A) selection kit, resuspend the mRNA capture beads in Swift RNA fragmentation buffer, following the Fragmentation on bead protocol below. See Appendix Section B for more detailed recommendations.

RNA Fragmentation •

Follow the appropriate fragmentation protocol below depending on the appropriate upstream workflow. Follow the fragmentation off bead protocol if purified mRNA or depleted/enriched RNA has been eluted in elution buffer, nuclease-free water, or another appropriate buffer. Follow the fragmentation on bead protocol if a poly(A) selection module is used and the RNA will go directly into the Swift RNA Library Kit following mRNA capture.

A fragmentation time of 10 min is recommended for high quality RNA (RIN ≥ 7) to achieve an insert size of 300-350 bp. See below for recommended fragmentation times for lower RIN scores or if a different insert size is desired. It is not recommended to prepare libraries from RNA with a RIN score < 2. See Appendix Section C for expected results using low-quality and FFPE RNA. See Appendix Section F for expected results using alternate fragmentation times with high-quality RNA.

Fragmentation off bead: compatible with purified mRNA or depleted/enriched RNA input following elution

1. On ice, bring RNA sample to a total volume of 5 µL in a 0.2 mL PCR tube, adding nuclease-free water if necessary.

2. Assemble the Fragmentation master mix on ice. Mix thoroughly and pulse spin to collect contents. Add 9 µL of the

mix below to each sample tube, mix thoroughly and pulse spin to collect contents (14 µL total reaction volume):

Component Volume per reaction

• RNA Reagent F1 1 µL


• RNA Buffer F3 4 µL


Volume to add 9 µL


3. Preheat thermocycler to 94 °C (Lid heating ON). Once it has reached 94 °C, add sample tubes to the thermocycler and run the program most appropriate for each sample:

RIN score Insert Size (bp) Temperature Time

≥7 300-350 94 °C 10 min

≥7 250-300 94 °C 12 min

≥7 200-250 94 °C 15 min

2-7 300-350 94 °C 5 min

FFPE 200-250 65 °C 5 min

4. When the program has complete, immediately transfer samples to ice and incubate for 2 min. Proceed directly to Reverse Transcription.

Fragmentation on bead: compatible with poly(A) selection modules

1. Assemble the Fragmentation master mix on ice. Mix thoroughly and pulse spin to collect contents (15 µL total reaction volume):




• RNA Buffer F3 4 µL


Nuclease-free water 6 µL

Volume to add 15 µL

2. Follow the poly(A) selection module protocol according to the manufacturer’s specifications (see Appendix Section B).

3. At the last step, do not resuspend the mRNA capture beads in elution buffer. Instead, resuspend the beads in 15

µL of the above Fragmentation master mix. Pipette up and down to mix thoroughly.

Safe stopping point – samples can be stored on-bead in the Fragmentation master mix at 4 °C for up to 24 hours. Do not freeze.

5. Preheat thermocycler to 94 °C. Once it has reached 94 °C, add sample tubes to the thermocycler and run the program most appropriate for each sample (Lid heating ON):

RIN score Insert Size (bp) Temperature Time

≥7 300-350 94 °C 10 min

≥7 250-300 94 °C 12 min

≥7 200-250 94 °C 15 min

2-7 300-350 94 °C 5 min

FFPE 200-250 65 °C 5 min

4. When the thermocycler program has completed, immediately place the tubes on a magnetic rack, wait for the

solution to clear, and transfer 14 µL of the supernatant to a new tube. Do not let the samples cool prior to placing on the magnetic rack. If cooled, mRNA fragments will re-hybridize to the mRNA capture beads.

5. Immediately transfer samples to ice and incubate for 2 min. Proceed directly to Reverse Transcription.


Reverse Transcription •

1. Assemble the Reverse Transcription master mix on ice. Mix thoroughly and pulse spin to collect contents. Add 6

µL of the mix below to each sample tube, mix thoroughly and pulse spin to collect contents (20 µL total reaction volume):


• RNA Enzyme R1 1 µL

• RNA Enzyme R2 1 µL

Nuclease-free water 4 µL

Volume to add 6 µL

2. Run the following thermocycler program (Lid heating ON):

Temperature Time

25 °C 10 min

42 °C 30 min

70 °C 15 min

4 °C hold

3. Proceed to SPRI clean-up.

SPRI Clean-up

Perform a SPRI clean-up following the ratio selected from the table below. See Appendix Section A for full SPRI clean-up protocol.

1. Add 30 µL Low EDTA TE to each sample (50 µL total volume).

2. Add SPRI beads according to the Bead Volume in the table below and follow clean-up protocol.

Insert Size (bp) SPRI ratio Bead Volume

300-350 1.0X 50 µL

250-300 1.4X 70 µL

200-250 1.8X 90 µL

RNA Sample SPRI ratio Bead Volume

FFPE 1.2X 60 µL

3. Elute in 12 µL Low EDTA TE and transfer 10 µL eluate to a fresh tube.

Safe stopping point – samples can be stored at 4 °C for up to 24 hours or at -20 °C for up to one month.


Adaptase •

1. Following elution from the SPRI clean-up step, there will be a 10 µL eluate in each tube.

2. Pre-assemble the Adaptase master mix on ice. Mix thoroughly and pulse spin to collect contents. Do NOT add to samples until after denaturation (Step 3). Keep master mix on ice until Step 5 below.


• RNA Buffer A1 2 µL

• RNA Reagent A2 2 µL

• RNA Reagent A3 1.25 µL

• RNA Enzyme A4 0.5 µL

• RNA Enzyme A5 0.5 µL

Low EDTA TE 4.25 µL

Volume to add 10.5 µL

3. Preheat thermocycler to 95 °C for denaturation of the samples. When the thermocycler has reached 95 °C, add

sample tubes (10 µL eluate) and run the following thermocycler program (Lid heating ON):

Temperature Time

95 °C 2 min

4. When the program has completed, immediately transfer samples to ice and incubate on ice for 2 min. Proceed directly to the next step.

5. Add 10.5 µL of the Adaptase master mix from Step 2 to each sample tube, mix thoroughly and pulse spin to

collect contents (20.5 µL total reaction volume).


Temperature Time

37 °C 15 min

95 °C 2 min

4 °C hold

7. Proceed to Extension.

Extension •

1. Assemble the Extension master mix on ice. Mix thoroughly and pulse spin to collect contents. Add 23 µL of the

mix to each sample tube, mix thoroughly and pulse spin to collect contents (43.5 µL total reaction volume):


• RNA Reagent E1 1 µL

• PCR Master Mix 22 µL



Temperature Time

98 °C 1 min

63 °C 2 min

72 °C 5 min

4 °C hold



SPRI Clean-up

Perform two (2) 1.2X SPRI clean-ups. See Appendix Section A for full SPRI clean-up protocol.

1. Add 52.2 µL SPRI beads to each sample and follow clean-up protocol.


3. Perform a second clean-up at a 1.2X ratio. Add 60 µL SPRI beads to each sample and follow clean-up protocol.



Ligation •

1. Assemble the Ligation master mix on ice. Mix thoroughly and pulse spin to collect contents. Add 15 µL of the mix

to each sample tube, mix thoroughly and pulse spin to collect contents (30 µL total reaction volume):


• RNA Buffer L1 3 µL

• RNA Reagent L2 10 µL

• RNA Enzyme L3 2 µL



Temperature Time

25 °C 15 min

4 °C hold


SPRI Clean-up

Perform a 1.0X SPRI clean-up before PCR. See Appendix Section A for full SPRI clean-up protocol.

1. Add 30 µL SPRI beads to each sample and follow clean-up protocol.



Indexing PCR •

1. Add 2.5 µL (of each combinatorial dual) or 5 µL (single or unique dual) of the appropriate indexing primer(s)

directly to each sample following the table below (25 µL total reaction volume). NOTE: If using Normalase Indexing Primers, see Appendix Section E and the Normalase Kit Protocol for specific instructions.

Indexing Kit* Reagent Reagent Contents Volume added to each sample

Single Indexing Index X Pre-mixed Universal and i7 primers 5 µL


D50X i5 primer 2.5 µL

D7XX / S7XX i7 primer 2.5 µL

Unique Dual Indexing U001-U096 Pre-mixed i5 and i7 primers 5 µL

*See Appendix Section J for the sequences corresponding to each Index.


IMPORTANT! Indexing primers are provided separately. Compatible Swift Indexing Kits include Cat. Nos. X6024, X8096, X85192, X86192, X87192, X88192, X89768, X9096, X90384, X9096-PLATE, X90384-PLATE, and X92034-PLATE.

2. Assemble the PCR master mix on ice. Mix thoroughly and pulse spin to collect contents. Add 25 µL of the mix to

each tube, mix thoroughly and pulse spin to collect contents (50 µL total reaction volume):




3. Run the following thermocycler program, adjusting the number of cycles depending on the input amount and sample quality (see table below) (Lid heating ON):

Temperature Time

98 °C 2 min

98 °C 20 sec

60 °C 30 sec

72 °C 30 sec

Perform X cycles*

72 °C 1 min

4 °C hold

* The recommended minimum number of cycles for each input in order to provide ≥4 nM yields suitable for direct sequencing is as follows:

mRNA/purified RNA input amount

Minimum number of cycles

Total RNA (before enrichment/selection)

Minimum number of cycles

100 pg 18 10 ng 17

1 ng 14 50 ng 14

10 ng 11 100 ng 13

100 ng 8 1 µg 8

The number of cycles required may vary based on the input amount, as detailed above, but also on the quality of the sample. Recommendations above are for high-quality input RNA (RIN ≥ 7). Low-quality and FFPE RNA may require additional cycles to reach a minimum yield of 4 nM.


SPRI Clean-up

Perform a 0.85X SPRI clean-up following the indexing PCR step. See Appendix Section A for full SPRI clean-up protocol.

1. Add 42.5 µL SPRI beads and follow clean-up protocol.


For low inputs (≤ 1 ng mRNA or ≤ 50 ng total RNA), adapter dimers may be present, evident by a peak at ~150 bp. For these samples, perform a second 0.85X SPRI on the samples prior to library quantification.

1. If necessary, add Low EDTA TE to sample to bring it to a total volume of 20 µL.

2. Add 17 µL SPRI beads and follow clean-up protocol.


Safe stopping point – samples can be stored at 4 °C for up to 24 hours or at -20 °C long-term. The library is now ready for quantification and sequencing.


Expected Results Expected yields for varying input quantities using the Swift RNA protocol with a starting input of human brain mRNA (Clontech 636102). Mean library size includes the adapter sequences which adds ~130 bp to the library insert size.

Input amount (mRNA) Mean library size (bp) PCR cycles Expected yield

100 pg 480 18 ≥4 nM

1 ng 480 14 ≥4 nM

100 ng 480 8 ≥4 nM

A peak at ~150 bp indicates adapter dimer carry-over. These can be removed with a second post-PCR SPRI (see page 12). Note that a peak around ~60 bp represents carryover of indexing primers from PCR and is expected. These fragments cannot cluster and will not impact quantification or sequencing but can be removed by a second post-PCR SPRI, if desired.

Library Quantification

Accurate library quantification is essential to properly loading the sequencing instrument. Libraries can be quantified using fluorometric-, electrophoretic-, or qPCR-based methods. Electrophoresis-based methods also allow examination of library insert size distribution. There are many commercially available kits suitable for library quantification.

Following the recommended PCR cycles will result in a library concentration of at least 4 nM.

Sequence the RNA Libraries

Libraries can be sequenced after accurate library quantification. Swift RNA libraries can be sequenced using single-end or paired-end sequencing on Illumina instruments. We recommend using a minimum of 2 x 50 paired-end reads. The depth of coverage required will depend on the application.

Due to the complexity of the transcriptome, no PhiX spike-in is required on MiSeq or MiniSeq instruments. The NextSeq550 may be sensitive to the low complexity Adaptase tail present at the beginning of Read 2 and PhiX or another suitable high-complexity library spike-in may be required. Contact Illumina technical support for further information regarding sequencing instrument compatibility with low-complexity sequences. If sequencing low-plex pooled single-indexed libraries, follow the recommendations outlined in the Illumina Index Adapters Pooling Guide. Use additional index combinations as needed for color-balanced index reads if required for the instrument.

IMPORTANT! To ensure optimal mapping efficiency, we recommend using the STAR aligner (Dobin et al. 2013). If using an alternate software for alignment, bioinformatic trimming of the low complexity Adaptase tail from these libraries may be required. See Appendix Section G.

Figure 2: Swift RNA Bioanalyzer Results. Expected results for Swift RNA libraries produced from starting inputs of 100 pg, 1 ng, or 100 ng human brain mRNA (Clontech 636102). Libraries were run on the Bioanalyzer using a HS DNA chip.


Appendix

Section A: SPRIselect Clean-Up Protocol

Please use the following protocol for each SPRI Step, substituting in the correct SPRI bead volume, and elution volume as indicated for each clean-up step. Perform all steps at room temperature.

1. Prepare a fresh 80% ethanol solution; prepare enough for ~3 mL per sample. 2. Invert or briefly vortex beads to homogenize the suspension before use. 3. Add the specified SPRI bead volume to each sample. Mix by pipetting 10 times or until homogenous. Ensure no

bead-sample suspension droplets are left on the sides of the tube. 4. Incubate the samples for 5 minutes at room temperature. 5. Pulse-spin the samples in a microfuge. Place the sample tubes on a magnetic rack until the solution clears and a

pellet is formed (~2 minutes). 6. Remove and discard the supernatant without disturbing the pellet.

7. Add 200 µl of freshly prepared 80% ethanol solution to the pellet while it is still on the magnet. Use care not to disturb the pellet. Incubate for 30 seconds, and then carefully remove the ethanol solution.

8. Repeat step 6 once for a second wash with the ethanol solution. 9. Pulse-spin the samples in a microfuge, place back onto the magnet and remove any residual ethanol solution

from the bottom of the tube. 10. Without delay to avoid over-drying of beads, add the specified elution volume of Low EDTA TE to resuspend the

pellet, mixing well by pipetting up and down until homogenous. If droplets of the resuspension are on the side of the tube, pulse-spin the tube in a microfuge to collect contents. After at least 2 minutes, place the tube on the magnetic rack and wait until the solution clears and a pellet is formed (~2 minutes).

11. Transfer the specified eluate volume to a new 0.2 mL PCR tube. Ensure that eluate does not contain magnetic beads (indicated by brown coloration in eluate). If magnetic beads are present, pipette eluate into a new tube, place on magnet, and transfer eluate again.

Section B: Kit Compatibility with Upstream Modules

The Swift RNA Library Kit is compatible with many poly(A) selection and ribosomal RNA depletion modules. See the below details for specific modules and recommendations on combining them with the Swift RNA Library Kit workflow. Please contact Swift Tech Support ([email protected]) for additional questions or recommendations for modules not listed below.

Poly(A) Selection Modules

Poly(A) selection allows for the enrichment of mRNA by capturing the poly(A) tail of transcripts using oligo-dT beads. We recommend using the following:

• NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB E7490S/L) - recommended

• Lexogen Poly(A) RNA Selection Kit (Lexogen 039.100) – alternate

If using the NEBNext Poly(A) mRNA Magnetic Isolation Module, follow the manufacturer’s specifications until the final elution (step 37 in the NEB Instruction Manual or the NEB Online Protocol, Version 6.0). Instead of resuspending the

mRNA capture beads in 17 µL of Tris Buffer, resuspend the mRNA capture beads in 15 µL of the Swift Fragmentation master mix and follow the Fragmentation on bead protocol on pages 7-8 of this document.

If using the Lexogen Poly(A) RNA Selection Kit, follow the manufacturer’s specifications until the final elution (step 12 in the Lexogen User Guide, Version 1.04). Completely remove the supernatant from the last wash. Instead of resuspending

the beads in 25 µL of Molecular Biology Grade Water, resuspend the beads in 15 µL of the Swift Fragmentation master mix and follow the Fragmentation on bead protocol on pages 7-8 of this document.

Ribosomal RNA Depletion Modules

Ribosomal RNA depletion allows for the depletion of ribosomal RNAs which make up 80-90% of total RNA. We recommend using the following:

• Lexogen RiboCop rRNA Depletion Kit V1.3 (Lexogen 037, 125-127) - recommended

• NEBNext rRNA Depletion Kit (NEB E6310 or E6350) - alternate


If using the Lexogen RiboCop rRNA Depletion Kit V1.3, follow the manufacturer’s specifications until the final elution (step

27 in the Lexogen Human/Mouse/Rat User Guide, step 22 in the Lexogen Bacteria User Guide). Instead of adding 12 µL

of Elution Buffer to the tube, add 7 µL of Elution Buffer to the tube. Remove the tube from magnet and resuspend the beads by pipetting up and down. Incubate for 2 minutes at room temperature. Place the tube on a magnetic rack. When

the solution clears, transfer 5 µL of the supernatant into a new PCR tube. Follow the Fragmentation off bead protocol as on pages 7-8 of this document.

If using the NEBNext rRNA Depletion Kit, follow the manufacturer’s specifications until the final elution (step 4.9 in the NEB Instruction Manual or the NEB Online Protocol, Version 5.0). Remove the tube/plate from the magnetic stand.

Instead of eluting the RNA in 8 µL of nuclease-free water, elute the RNA in 7 µL of nuclease-free water. Mix well by pipetting up and down 10 times. Incubate for at least 2 minutes. Place the tube/plate on the magnetic stand. When the

solution is clear, transfer 5 µL to a new PCR tube. Follow the Fragmentation off bead protocol on pages 7-8 of this document.

Section C: Low-quality and FFPE RNA

The quality of input RNA into an RNA-Seq workflow can substantially impact the library yield and resulting data quality. RNA quality and integrity can be evaluated using two different metrics: RIN score (RNA Integrity Number; ratio of the 28S to 18S rRNA peaks) and DV200 (percentage of RNA fragments that are greater than 200 nucleotides). Both of these metrics can be obtained using an electrophoretic instrument, such as an Agilent Bioanalyzer or Tapestation. High-quality samples typically have RIN scores ≥ 7 and are suitable for poly(A)-selection. Samples with RIN scores between 2 and 7 are suitable for ribodepletion or hybridization capture. For these samples, more information about sample quality can be obtained through the DV200 score which can help inform the fragmentation time, SPRI ratio, and PCR cycling. In general, it is not recommended to make libraries using samples with a RIN < 2 or a DV200 < 30.

Expected Results with Low-quality RNA (RIN < 7)

Universal Human Reference (UHR) RNA (Agilent 740000) was heat-damaged according to the table below to produce variable RIN scores. RIN scores were evaluated using an RNA Pico 6000 Bioanalyzer Kit (see below).

RIN Score Heat-treatment temperature Heat-treatment time

8.8 NA NA

5.8 80 °C 5 min

3.6 80 °C 8 min

2.0 80 °C 12 min

Figure 3: Bioanalyzer Results for Heat-damaged UHR. RNA Bioanalyzer results for heat-damaged UHR to obtain various RIN scores. UHR RNA samples were run on the Bioanalyzer using an RNA Pico 6000 chip. The Total Eukaryote RNA program was run in order to obtain RIN score estimates.


Expected library results using 100 ng UHR with RIN scores of 2.0, 3.6, 5.8, and 8.8 as input into the Swift RNA Library Kit. Fragmentation times were adjusted as recommended (see pages 7-8 and the table below) with 8 PCR cycles used for each library.

RIN Score Fragmentation Time Mean library size (bp) Mean insert size (bp) Expected yield

8.8 10 min 480 350 ≥4 nM

5.8 5 min 480 350 ≥4 nM

3.6 5 min 465 335 ≥4 nM

2.0 5 min 450 320 ≥4 nM

Expected Results with FFPE RNA (RIN ~2, DV200 < 80)

FFPE curls were obtained from breast cancer tumor samples (Spectrum Health; Grand Rapids, MI). FFPE RNA was

extracted using the RNeasyâ FFPE Kit (Qiagen 73504). RIN and DV200 scores were evaluated using an RNA Pico 6000 Bioanalyzer Kit (see below). Trace analyses show the samples have RIN scores of ~2 and DV200 scores of 48 and 73.

Because FFPE RNA samples typically have low integrity (RIN < 7), the recommended NGS workflows are ribodepletion or hybridization capture. For ribodepletion, see Appendix Section B for instructions on pairing the Swift RNA kit with compatible ribodepletion modules. For hybridization capture, see Appendix Section D for recommended protocol adjustments.

Following ribodepletion, FFPE-specific adjustments to the Swift RNA protocol are as follows:

• Fragmentation: Instead of fragmenting samples at 94 °C, heat samples at 65 °C for 5 minutes (see page 8). This step may require sample-specific optimization; reduce fragmentation time by 1-2 min for low DV200 scores (DV200 < 40) or add 1-2 min for high DV200 scores (DV200 > 70).

• Post-RT SPRI ratio: Instead of using a 1.0X SPRI ratio in the clean-up following reverse transcription, use a 1.2X SPRI ratio (see page 9). To achieve a 1.2X ratio, add 60 µL of SPRI beads to the 50 µL sample volume. This step may require sample-specific optimization; increase the SPRI ratio up to 1.8X to retain smaller fragments for low DV200 scores (DV200 < 40).

Expected library results using 100 ng FFPE RNA into the Lexogen Ribocop V1.3 ribodepletion module. Ribodepleted RNA was used as input into the Swift RNA Library Kit with the following adjustments: fragmentation at 65 °C for 5 min and 1.2X post-RT SPRI clean-ups. Libraries were amplified with 12 PCR cycles.

Sample RIN

Score DV200 Score

Fragmentation Time

Post-RT SPRI

Mean library size (bp)

Mean insert size (bp)

Library Yield

FFPE Sample 1 2.1 48 5 min @ 65 °C 1.2X 400 270 20 nM

FFPE Sample 2 2.0 73 5 min @ 65 °C 1.2X 470 340 24 nM

DV200 = 48

RIN = 2.1

DV200 = 73

RIN = 2.0

Figure 4: Bioanalyzer Results for FFPE RNA. RNA was isolated from breast cancer tumor samples. RNA samples were run on the Bioanalyzer using an RNA Pico 6000 chip. The Total Eukaryote RNA program was run in order to obtain RIN scores. The DV200 RNA Pico program was used to obtain DV200 scores.


For more information, see the Swift Biosciences Application Note titled “The Swift RNA Library Kit Optimizes RNA-Seq Data Quality and Costs for FFPE Samples.”

Section D: Protocol Adjustments for Hybridization Capture Hybridization capture enables the capture and sequencing of specific regions of the transcriptome. First, libraries are made using total RNA. Next, a probe set designed to a target region (such as the exome) is used to hybridize and capture complementary fragments. The captured library is then sequenced. Hybridization capture is compatible with intact as well as damaged or degraded samples, such as FFPE RNA.

The Swift RNA Library Kit is compatible with the Swift Hyb, Wash, and Universal Blocker Kit (Cat. No. 89016) and all Swift Hyb Panels (Exome, Cat. No. 83216; Pan-Cancer, Cat. No. 83316; Inherited Diseases, Cat. No. 83416). If using a hybridization capture kit from a different supplier, contact Swift Tech Support ([email protected]) to discuss compatibility and indexing primer strategies.

Hybridization capture requires small insert sizes (~200 bp) and high yields (~200 ng per library is required for most RNA capture protocols). In order to meet these requirements, follow the below protocol adjustments:

• Fragmentation time: For intact RNA, fragment samples at 94 °C for 15 min; for FFPE RNA, fragment samples at 94 °C for 2 min. This step may require sample-specific optimization; reduce fragmentation time if library insert sizes are <150 bp or increase fragmentation time if library insert sizes are >200 bp.

• SPRI ratio: To accommodate smaller insert sizes, adjust the SPRI ratio of ALL clean-ups to 1.8X. For each clean-up, multiply the sample volume by 1.8 to determine the volume of SPRI beads to add (for example, add 90 µL of SPRI beads to the 50 µL sample volume following reverse transcription).

• PCR cycles: To achieve >200 ng library yields, add a minimum of 4 PCR cycles to the mRNA/purified RNA recommended number of cycles that matches your input amount. For example, for 100 ng of total RNA, use 12 PCR cycles (where 8 is the recommended number of cycles for 100 ng mRNA/purified RNA; see page 12). This step may require sample-specific optimization; increase the number of PCR cycles if yields are lower than the required amount for hybridization capture.

Expected library results using 100 ng FFPE RNA into the Swift RNA Library Kit with the following adjustments: fragmentation at 94 °C for 2 min and 1.8X post-RT SPRI clean-ups. Libraries were amplified with 12 PCR cycles.

Sample RIN

Score DV200 Score

Fragmentation Time

All SPRI Mean library

size (bp) Mean insert

size (bp) Library Yield

FFPE Sample 1 2.1 48 2 min @ 94 C 1.8X 300 170 1.4 µg

FFPE Sample 2 2.0 73 2 min @ 94 C 1.8X 330 200 1.8 µg

Figure 5: Bioanalyzer Results for Libraries from Ribodepleted FFPE RNA. Expected results for Swift RNA libraries produced from 100 ng FFPE RNA following ribodepletion using Lexogen RiboCop v1.3. Libraries were run on the Bioanalyzer using a HS DNA chip.


Following preparation of the libraries from total RNA using the Swift RNA Library Kit, 200 ng of each library was pooled and used as input into the Swift Exome Hyb Panel (Cat. No. 83216). Captured libraries were amplified with 7 post-hyb PCR cycles. Expected results for a multiplexed hybridization capture library are detailed in the table below.

Sample Number of

multiplexed samples* Post-Hyb PCR

cycles Mean library

size (bp) Mean insert

size (bp) Library Yield

Hybridization Capture Library

2 7 330 200 20 nM

*Note: For RNA-Seq libraries, we do not recommend multiplexing more than 6 samples using the Swift Hyb, Wash, and Blocker kit paired with a Swift Hyb Panel. If using a hybridization capture kit from a different supplier, please follow the recommendations provided by that supplier.

For more information, see the Swift Biosciences Application Note titled “The Swift RNA Library Kit Optimizes RNA-Seq Data Quality and Costs for FFPE Samples.”

Section E: Normalase Instructions

Please review this section and the Normalase Kit protocol before setting up your indexing PCR. In order to achieve expected results, amplify each library using Normalase indexing primers (Cat No. 68096) with the appropriate number of

cycles and thermocycling conditions below to obtain a library yield of 12 nM or greater in a 20 µL eluate.

1. Add 2 µL of each Normalase combinatorial dual index primer to each sample (24 µL total volume).

Normalase Reagent Volume added to each sample

D50XN 2 µL

D7XXN 2 µL

*See Appendix Section J for the sequences corresponding to each Index.

Figure 6: Bioanalyzer Results for Libraries intended for Hybridization Capture. Expected results for Swift RNA libraries produced from 100 ng total FFPE RNA. The following protocol adjustments were implemented:

fragmentation at 94 °C for 2 min, 1.8X SPRI ratio for all clean-ups, 12 PCR cycles. Libraries were diluted 1:10 prior to running on the Bioanalyzer using a HS DNA chip.


2. Assemble the PCR master mix on ice. Mix thoroughly and pulse spin to collect contents. Add 26 µL of the mix to

each sample tube, mix thoroughly and pulse spin to collect contents (50 µL total reaction volume):



• Reagent R6 1 µL


3. Run the following thermocycler program, adjusting the number of cycles depending on the input amount and sample quality (see table below) (Lid heating ON):

Temperature Time

98 °C 2 min

98 °C 20 sec

60 °C 30 sec

72 °C 30 sec

Perform X cycles*

72 °C 5 min

4 °C hold

* The recommended minimum number of cycles for each input in order to provide ≥12 nM yields suitable for the Normalase workflow is as follows:

mRNA/purified RNA input amount

Minimum number of cycles for ≥12 nM

Total RNA (before enrichment/selection)

Minimum number of cycles for ≥12 nM

100 pg 20 10 ng 20

1 ng 17 50 ng 17

100 ng 9 1 µg 9

The number of cycles required may vary based on the input amount, as detailed above, but also on the quality of the sample. Recommendations above are for high-quality input RNA (RIN ≥ 7).

4. Proceed to post-PCR SPRI clean-up (page 12 of this protocol).

5. Proceed to Normalase I, Pooling, and Normalase II in the Normalase Kit Protocol.

Section F: Expected Results for Alternate Insert Sizes

To adjust the final mean library size, adjust both the fragmentation time and SPRI ratio following reverse transcription. Increase both the fragmentation time and post-RT SPRI ratio to achieve a smaller insert size.

Expected library results using 100 ng human brain mRNA into the Swift RNA Library Kit with fragmentation time and post-RT SPRI ratio adjustments are detailed in the table below. Libraries were amplified with 8 PCR cycles.

Fragmentation Time Post-RT SPRI Ratio Mean library size (bp) Mean insert size (bp) Expected yield

10 min (default) 1.0X 480 350 ≥4 nM

12 min 1.4X 430 300 ≥4 nM

15 min 1.8X 380 250 ≥4 nM


Section G: Data Analysis and Informatics

Swift Biosciences’ Adaptase technology, used in the Swift RNA Library Kit, adds a low-complexity polynucleotide tail with a median length of 8 bases to the 3’ end of each fragment during the addition of the second NGS adapter molecule. Therefore, it is normal and expected to observe this tail at the beginning of Read 2 (R2). When read length is close to fragment size, the tail may also be observed toward the end of Read 1 (R1) data.

We recommend using the STAR aligner (Dobin et al. 2013) as it is typically able to soft clip the synthetic Adaptase tail sequence as well as the synthetic random primer sequence at the beginning of Read 1 if any mismatches were introduced during the priming step. STAR provides efficient mapping without additional processing of the sequencing data. However, if you find that soft-clipping is not sufficient for your particular analysis, we recommend implementing STAR with the following argument: --clip5pNbases 10

Other aligners may be unable to soft clip the synthetic tail sequence which can interfere with alignment. In most cases, a reciprocal trim is preferred. We recommend trimming 15 bases from the beginning of both R1 and R2 if insert size is significantly larger than read length (i.e., 2 x 75 bp for a 250 bp insert library). If insert size is approaching the read length, you may encounter tails at the end of R1 (i.e., 2 x 125 bp for a 250 bp library). We recommend that you instead trim 15 bases from the end of R1 and the beginning of R2. Tail and random primer trimming can be performed using publicly available tools like Trimmomatic (Bolger, et al. 2014 Bioinformatics) or Cutadapt (Martion, et al. 2011 EMBnet.journal).

Ensure that tail trimming is performed AFTER adapter trimming.

For additional tail trimming recommendations, please consult our Technical Note titled “Accel-NGS 1S Plus and Methyl-Seq: Tail Trimming for Better Data” or contact Swift at [email protected].

Section H: Notes for Automation

This protocol is readily automatable. A 10% overage volume of reagents is supplied to accommodate automation. Please contact us at [email protected] if you require additional reagent overage volume or would like to learn about our custom packaging options.

While Swift Biosciences does not supply automated liquid handling instruments or consumables, our automation team collaborates with automation solution providers and customers to develop and qualify optimized automated scripts for use of our kits with liquid handling platforms routinely used in NGS library preparation. Please contact us at [email protected] to discuss automating the Swift RNA Library Kit with your particular automated liquid handling system.

Figure 7: Swift RNA Bioanalyzer Results for Alternate Insert Sizes. Expected results for Swift RNA libraries produced from starting human brain mRNA (Clontech 636102) inputs of 100 ng using a fragmentation time of 10, 12, or 15 minutes with post-RT SPRI ratios of 1.0X, 1.4X, and 1.8X, respectively. Libraries were run on the Bioanalyzer using a HS DNA chip.


Section I: Troubleshooting Common Problems

Problem Possible Cause Suggested Remedy

Input is less than 100 pg

of RNA in 5 µL volume.

Input RNA is too dilute. Concentrate RNA with column purification kit, SpeedVac, or other method.

Difficulty resuspending beads after ethanol wash during SPRI steps.

Over-drying of beads. Add Low EDTA TE immediately after removing the final ethanol wash. Continue pipetting the liquid over the beads for complete resuspension.

Lower than expected cluster density.

Error in library quantification. Re-quantify library and confirm correct library insert size for calculating molarity.

Additional peak around ~150 bp visible on Bioanalyzer trace.

Truncated adapters were carried over into the Ligation reaction, resulting in adapter dimers.

Perform an additional 0.85X SPRI on samples to remove residual adapter dimers prior to quantifying or sequencing. Note that a peak around ~60 bp represents carryover of indexing primers from PCR and is expected. These fragments cannot cluster and will not impact quantification or sequencing.

Library size (insert size plus ~130 bp to account for adapters) is much smaller or larger than the expected

Too much or too little fragmentation, or sample had different RIN score than anticipated.

Adjust the fragmentation time to optimize the library size to fit your experimental requirements. Increase fragmentation by ~2 minutes for a smaller library size or decrease by ~2 minutes for a larger library size. See Appendix Section F for more details.

Yields are lower than expected (< 4 nM)

Inaccurate RNA input quantification, low-quality RNA, or incorrect SPRI ratios

Ensure you are accurately quantifying your RNA input with Nanodrop, Qubit, or Bioanalyzer prior to going into the Swift RNA Library Kit. If using low-quality RNA (RIN <7), see our recommendations in Appendix Section C. PCR cycles may need to be increased to accommodate your specific RNA input if yields are consistently lower than expected.

A second, large peak is visible in the Bioanalyzer trace (> 2000 bp)

Libraries were overamplified, resulting in the formation of a heteroduplex

Reduce the number of PCR cycles to prevent the overamplification of your libraries.

If you experience problems with your library prep, please contact Swift at [email protected], or by phone: 734.330.2568 (9:00 am – 5:00 pm ET, Monday through Friday).


Section J: Indexed Adapter Sequences

During the Indexing PCR step in the protocol, you must use a unique indexing primer Index X (single index), D50X and D7XX/S7XX (combinatorial dual indexes), or U001-U096 (unique dual indexes) to label each library. If no multiplex sequencing is being performed, all libraries may be labeled with a single index only. Libraries made with uniquely indexed adapters may be pooled prior to cluster generation and co-sequenced on the same Illumina flow cell. The full-length adapter sequences of the single, combinatorial dual, and unique dual indices are below. The underlined text indicates the location of the index sequences, as detailed in the tables below.

Single Indexing and Unique Dual Indexing primer kits provide the appropriate Universal or i5 and i7 primers mixed

together in each tube. During the Indexing PCR step, 5 µL of the primer set is required for each reaction. Combinatorial Dual and Normalase Combinatorial Dual Indexing primer kits provide the i5 and i7 primers in separate tubes. During the

Indexing PCR step, 2.5 µL or 2 µL of the selected i5 primer and 2.5 µL or 2 µL of the selected i7 primer is required for each reaction, for Combinatorial Dual and Normalase Combinatorial Dual Indexing, respectively. Please reference the table below and see Indexing PCR on page 10 or Normalase instructions on page 16 of this protocol.

Indexing Kit Reagent Reagent Contents Volume added to

each sample

Single Indexing Index X Pre-mixed Universal and i7 primers 5 µL


D50X i5 primer 2.5 µL

D7XX / S7XX i7 primer 2.5 µL

Normalase Combinatorial Dual Indexing

D50XN i5 primer 2 µL

D7XXN i7 primer 2 µL

Unique Dual Indexing U001-U096 Pre-mixed i5 and i7 primers 5 µL

Single Indexing (Set A Indexing Kit, Cat. No. X6024)

TruSeq Universal Adapter:

5’ AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT

TruSeq Index Adapter (I2, I4, I5, I6, I7, I12):

5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCACXXXXXX(AT)CTCGTATGCCGTCTTCTGCTTG

TruSeq Index Adapter (I13, I14, I15, I16, I18, I19):

5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCACXXXXXX(XX)ATCTCGTATGCCGTCTTCTGCTTG

The number on the product tube label indicates which indexing primer is provided in the tube. The bases in parentheses are not considered part of the 6-base index sequences but can be used for 8-base index reads.

Each tube contains 11 µL of the appropriate indexing primer set.

Set A Adapter i7 Sequence Set A Adapter i7 Sequence

Index 2 CGATGT(AT) Index 13 AGTCAA(CA)

Index 4 TGACCA(AT) Index 14 AGTTCC(GT)

Index 5 ACAGTG(AT) Index 15 ATGTCA(GA)

Index 6 GCCAAT(AT) Index 16 CCGTCC(CG)

Index 7 CAGATC(AT) Index 18 GTCCGC(AC)

Index 12 CTTGTA(AT) Index 19 GTGAAA(CG)


Combinatorial Dual Indexing (CD Indexing Kit, Cat. No. X8096; Swift S1-S4 Indexing Kit, Cat. Nos. X85192, X86192, X87192, X88192, X89768) and Normalase Combinatorial Dual Indexing Kit (Cat. No. 68096)

TruSeq Index 1 (i7) Adapter (D701-D712):

5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCACXXXXXXXXATCTCGTATGCCGTCTTCTGCTTG

TruSeq Index 2 (i5) Adapter (D501-D508):

5’ AATGATACGGCGACCACCGAGATCTACACXXXXXXXXACACTCTTTCCCTACACGACGCTCTTCCGATCT

For 96-plex Combinatorial Dual Indexing (Cat. No. X8096), each D50X tube contains 33 µL and each D7XX tube contains

22 µL of the appropriate indexing primer.

For 96-plex Normalase Combinatorial Dual Indexing (Cat. No. 68096), each D50XN tube contains 27 µL and each D7XXN

tube contains 18 µL of the appropriate indexing primer. The Reagent R6 tube contains 106 µL.

*Index sequences apply to both Combinatorial Dual and Normalase Combinatorial Dual Indexing Primer Kits. Normalase Combinatorial Dual Indexing Primers will be denoted as D50XN or D7XXN.

For 192-plex Combinatorial Dual Indexing (Cat. Nos. X85192, X86192, X87192, X88192), each D50X tube contains 66 µL

and each S7XX tube contains 22 µL of the appropriate indexing primer. For 768-plex Combinatorial Dual Indexing (Cat.

No. X89768), each D50X tube contains 300 µL and each S7XX tube contains 22 µL of the appropriate indexing primer.

Set S1 Index #

i7 Index Sequence

Set S2 Index #

i7 Index Sequence

Set S3 Index #

i7 Index Sequence

Set S4 Index #

i7 Index Sequence

S701 CAACACAG S725 GCTTCACA S749 TCCAGTCG S773 AGTCTGTA

S702 ACACCTCA S726 CGATGTTT S750 TGTATGCG S774 CCGTATCT

S703 ACCATAGG S727 TTAGGCAT S751 TCATTGAG S775 CGCTTCCT

S704 CAGGTAAG S728 ACAGTGGT S752 TGGCTCAG S776 CAAGACCT

S705 AACGCACA S729 GCCAATGT S753 TATGCCAG S777 CCTAGTAT

S706 TAGTCTCG S730 CAGATCTG S754 TCAGATTC S778 CCACCGAT

S707 CAGTCACA S731 ACTTGATG S755 GGTTGGAC S779 CTATCATG

S708 CCAACACT S732 TAGCTTGT S756 GACACTTA S780 CATGAATG

Dual Index Adapter*

i5 Sequence for MiSeq, NovaSeq, HiSeq 2500

i5 Sequence for MiniSeq, NextSeq, HiSeq 4000

Dual Index Adapter*

i7 Sequence

Index D501 TATAGCCT AGGCTATA Index D701 ATTACTCG

Index D502 ATAGAGGC GCCTCTAT Index D702 TCCGGAGA

Index D503 CCTATCCT AGGATAGG Index D703 CGCTCATT

Index D504 GGCTCTGA TCAGAGCC Index D704 GAGATTCC

Index D505 AGGCGAAG CTTCGCCT Index D705 ATTCAGAA

Index D506 TAATCTTA TAAGATTA Index D706 GAATTCGT

Index D507 CAGGACGT ACGTCCTG Index D707 CTGAAGCT

Index D508 GTACTGAC GTCAGTAC Index D708 TAATGCGC

Index D709 CGGCTATG

Index D710 TCCGCGAA

Index D711 TCTCGCGC

Index D712 AGCGATAG


S709 ACATGCCA S733 TGGTTGTT S757 GCTATGGA S781 CTGTACGG

S710 ATTCCGCT S734 TGTACCTT S758 GTAACCGA S782 CACTCGAG

S711 CAAGGTAC S735 TCTGCTGT S759 GGCAAGCA S783 CCGACAAG

S712 CCATGAAC S736 TTGGAGGT S760 GAACGACA S784 CTTGCTTC

S713 TCAGCCTT S737 TCGAGCGT S761 GCGTCGAA S785 CGCCTTAT

S714 CAGTGCTT S738 TGATACGT S762 AAGGCGAT S786 GCAACCAT

S715 CTCGAACA S739 TGCATAGT S763 CAGGCATT S787 TGACCGTT

S716 ACAGTTCG S740 TGCGATCT S764 AACTGTAT S788 TTGAGCTC

S717 ATCCTTCC S741 TTCCTGCT S765 ATGCTTGA S789 CCACATTG

S718 CGAAGTCA S742 TACAGGAT S766 AGTATCTG S790 AGCCAACT

S719 CTCTATCG S743 TGTGGTTG S767 ATGTAATG S791 ATCACGTT

S720 ACTCTCCA S744 TTCCATTG S768 ACACATGT S792 TCTCGGTT

S721 TCCTCATG S745 TAACGCTG S769 ATAGCACG S793 TTGACTCT

S722 AACAACCG S746 TTGGTATG S770 ATATTGTA S794 TCGAAGTG

S723 CTCGTTCT S747 TGAACTGG S771 CAATTGAT S795 CACCCAAA

S724 TCAGTAGG S748 TACTTCGG S772 CACGTCGT S796 CTTCACAT

Swift Unique Dual Indexing (UDI Kit, Cat. No. X9096, X90384, X9096-PLATE, X90384-PLATE, & X92304-PLATE)

TruSeq Index 1 (i7) Adapters:

5’ – GATCGGAAGAGCACACGTCTGAACTCCAGTCACXXXXXXXXATCTCGTATGCCGTCTTCTGCTTG – 3’

TruSeq Index 2 (i5) Adapters:

5’ – AATGATACGGCGACCACCGAGATCTACACXXXXXXXXACACTCTTTCCCTACACGACGCTCTTCCGATCT – 3’

Each U0XX tube contains 22 µL of the appropriate indexing primer set.

UDI # i7 Index

Sequence

i5 Index Sequence for

NovaSeq, MiSeq, HiSeq

2500

i5 Index Sequence for HiSeq4000, NextSeq, MiniSeq

UDI # i7 Index

Sequence

i5 Index Sequence for

NovaSeq, MiSeq, HiSeq

2500

i5 Index Sequence for HiSeq4000, NextSeq, MiniSeq

U001 CAACACAG CTTCACAT ATGTGAAG U049 TCCAGTCG TACTTCGG CCGAAGTA

U002 ACACCTCA CACCCAAA TTTGGGTG U050 TGTATGCG TGAACTGG CCAGTTCA

U003 ACCATAGG TCGAAGTG CACTTCGA U051 TCATTGAG TTGGTATG CATACCAA

U004 CAGGTAAG TTGACTCT AGAGTCAA

U052 TGGCTCAG TAACGCTG CAGCGTTA

U005 AACGCACA TCTCGGTT AACCGAGA U053 TATGCCAG TTCCATTG CAATGGAA

U006 TAGTCTCG ATCACGTT AACGTGAT U054 TCAGATTC TGTGGTTG CAACCACA

U007 CAGTCACA AGCCAACT AGTTGGCT U055 GGTTGGAC TACAGGAT ATCCTGTA

U008 CCAACACT CCACATTG CAATGTGG

U056 GACACTTA TTCCTGCT AGCAGGAA

U009 ACATGCCA TTGAGCTC GAGCTCAA U057 GCTATGGA TGCGATCT AGATCGCA

U010 ATTCCGCT TGACCGTT AACGGTCA U058 GTAACCGA TGCATAGT ACTATGCA

U011 CAAGGTAC GCAACCAT ATGGTTGC U059 GGCAAGCA TGATACGT ACGTATCA

U012 CCATGAAC CGCCTTAT ATAAGGCG

U060 GAACGACA TCGAGCGT ACGCTCGA


U013 TCAGCCTT CTTGCTTC GAAGCAAG U061 GCGTCGAA TTGGAGGT ACCTCCAA

U014 CAGTGCTT CCGACAAG CTTGTCGG

U062 AAGGCGAT TCTGCTGT ACAGCAGA

U015 CTCGAACA CACTCGAG CTCGAGTG

U063 CAGGCATT TGTACCTT AAGGTACA

U016 ACAGTTCG CTGTACGG CCGTACAG U064 AACTGTAT TGGTTGTT AACAACCA

U017 ATCCTTCC CATGAATG CATTCATG U065 ATGCTTGA TAGCTTGT ACAAGCTA

U018 CGAAGTCA CTATCATG CATGATAG

U066 AGTATCTG ACTTGATG CATCAAGT

U019 CTCTATCG CCACCGAT ATCGGTGG

U067 ATGTAATG CAGATCTG CAGATCTG

U020 ACTCTCCA CCTAGTAT ATACTAGG U068 ACACATGT GCCAATGT ACATTGGC

U021 TCCTCATG CAAGACCT AGGTCTTG U069 ATAGCACG ACAGTGGT ACCACTGT

U022 AACAACCG CGCTTCCT AGGAAGCG

U070 ATATTGTA TTAGGCAT ATGCCTAA

U023 CTCGTTCT CCGTATCT AGATACGG

U071 CAATTGAT CGATGTTT AAACATCG

U024 TCAGTAGG AGTCTGTA TACAGACT U072 CACGTCGT GCTTCACA TGTGAAGC

U025 GCTTCACA CACGTCGT ACGACGTG U073 AGTCTGTA TCAGTAGG CCTACTGA

U026 CGATGTTT CAATTGAT ATCAATTG

U074 CCGTATCT CTCGTTCT AGAACGAG

U027 TTAGGCAT ATATTGTA TACAATAT

U075 CGCTTCCT AACAACCG CGGTTGTT

U028 ACAGTGGT ATAGCACG CGTGCTAT U076 CAAGACCT TCCTCATG CATGAGGA

U029 GCCAATGT ACACATGT ACATGTGT U077 CCTAGTAT ACTCTCCA TGGAGAGT

U030 CAGATCTG ATGTAATG CATTACAT

U078 CCACCGAT CTCTATCG CGATAGAG

U031 ACTTGATG AGTATCTG CAGATACT

U079 CTATCATG CGAAGTCA TGACTTCG

U032 TAGCTTGT ATGCTTGA TCAAGCAT U080 CATGAATG ATCCTTCC GGAAGGAT

U033 TGGTTGTT AACTGTAT ATACAGTT U081 CTGTACGG ACAGTTCG CGAACTGT

U034 TGTACCTT CAGGCATT AATGCCTG

U082 CACTCGAG CTCGAACA TGTTCGAG

U035 TCTGCTGT AAGGCGAT ATCGCCTT

U083 CCGACAAG CAGTGCTT AAGCACTG

U036 TTGGAGGT GCGTCGAA TTCGACGC U084 CTTGCTTC TCAGCCTT AAGGCTGA

U037 TCGAGCGT GAACGACA TGTCGTTC U085 CGCCTTAT CCATGAAC GTTCATGG

U038 TGATACGT GGCAAGCA TGCTTGCC

U086 GCAACCAT CAAGGTAC GTACCTTG

U039 TGCATAGT GTAACCGA TCGGTTAC

U087 TGACCGTT ATTCCGCT AGCGGAAT

U040 TGCGATCT GCTATGGA TCCATAGC U088 TTGAGCTC ACATGCCA TGGCATGT

U041 TTCCTGCT GACACTTA TAAGTGTC U089 CCACATTG CCAACACT AGTGTTGG

U042 TACAGGAT GGTTGGAC GTCCAACC

U090 AGCCAACT CAGTCACA TGTGACTG

U043 TGTGGTTG TCAGATTC GAATCTGA

U091 ATCACGTT TAGTCTCG CGAGACTA

U044 TTCCATTG TATGCCAG CTGGCATA U092 TCTCGGTT AACGCACA TGTGCGTT

U045 TAACGCTG TGGCTCAG CTGAGCCA U093 TTGACTCT CAGGTAAG CTTACCTG

U046 TTGGTATG TCATTGAG CTCAATGA

U094 TCGAAGTG ACCATAGG CCTATGGT

U047 TGAACTGG TGTATGCG CGCATACA

U095 CACCCAAA ACACCTCA TGAGGTGT

U048 TACTTCGG TCCAGTCG CGACTGGA U096 CTTCACAT CAACACAG CTGTGTTG

During library prep, make sure to note which indexing primer(s) is used with each sample and do not use the same indexing primer set on two different samples that will be multiplexed together.


Revision History Document # Revision Date Description of Change

PRT-023 Version 1.0 9/4/2019 Initial release.

PRT-023 Version 2.0 11/12/2019 Added Normalase compatibility.

PRT-023 Version 3.0 4/222020 Added FFPE and hybridization capture recommendations.

General Warranty

Swift Biosciences, Inc. (“Swift”) warrants that its products meet Swift’s specifications at the time of delivery. 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, implied or otherwise) as to quality, condition, description, merchantability, fitness for purpose, or non-infringement (except for the implied warranty of title) are hereby expressly excluded. All warranty claims on products must be made in writing within ninety (90) days of receipt of the products. Swift’s sole 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 Liability

Swift Biosciences, Inc. (“Swift”) shall have no liability under the warranties cited above with respect to any defect in the products arising from: (i) specifications or materials supplied by the buyer; (ii) willful damage or negligence of the buyer or its employees or agents; (iii) abnormal working conditions at the buyer’s premises; (iv) failure to follow Swift’s use restrictions 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 limitation, 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 License

This 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.

674 S. Wagner Road • Ann Arbor, MI 48103 • 734.330.2568 • www.swiftbiosci.com

© 2020, Swift Biosciences, Inc. The Swift logo and Swift Normalase are trademarks and Adaptase is a registered trademark of Swift Biosciences. Swift RNA Library Kit is covered by one or more claims of US Patent No(s). 9,896,709. This product is for Research Use Only. Not for use in diagnostic procedures. Illumina, MiniSeq, MiSeq, NextSeq, NovaSeq, HiSeq, and TruSeq are registered trademarks of Illumina, Inc. SPRI, SPRIPlate, and SPRIselect are trademarks of Beckman Coulter, Inc. NanoDrop and Qubit are registered trademarks and DynaMag is a trademark of Thermo Fisher Scientific, Inv. RNaseZap is a registered trademark of Applied Biosystems, Inc. NEBNext is a registered trademark of New England BioLabs, Inc. RiboCop is a trademark of Lexogen, GmbH. RNeasy is a registered trademark of QIAGEN. Oligonucleotide adapter sequences are copyrighted © 2019 Illumina, Inc. All rights reserved.

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