FAQs

The following frequently asked questions apply to all of our technology.

Our transposase-based plexWell technology offers simplicity, speed, and normalization without sacrificing quality. Download the User Guide for more FAQs and information here.

What is the best way to contact customer support?

Please email [email protected] or call the main seqWell number, +1-855-737-9355 and follow the prompts for support.

Are all required adapters, indices, and amplification primers included in the plexWell library preparation kits?

Yes. All plexWell kits include the indexed adapters, purification beads, and amplification primers necessary to make dual-indexed Illumina-compatible libraries.

Are any additional reagents, consumables, or equipment needed?

Please see the individual user guides for a complete list of required reagents, consumables, and equipment.

Where can I find the index sequences?

A complete list of all indices used in plexWell kits can be found in the index list under the resources section on the product page.

How long are the plexWell adapters?

The combined length of adapters and indexes are 135 bp.

Can I use a different polymerase for library amplification?

KAPA HiFi HotStart ReadyMix is the only DNA polymerase that has been validated with plexWell library preparation kits.  Contact [email protected] if you are unable to obtain the KAPA HiFI HotStart ReadyMix.

Do I need special sequencing primers or sequencing reagents?

plexWell libraries are compatible with the iSeq, MiniSeq, MiSeq, NextSeq, HiSeq, and NovaSeq sequencing systems. However, for older HiSeq models, the sequencing primers provided in TruSeq v3 Cluster kits are incompatible with Nextera-style libraries, including plexWell libraries. Consequently, the TruSeq Dual Index Sequencing Primer Box from Illumina is required for sequencing plexWell libraries on older systems, such as the HiSeq 2500, HiSeq 2000, HiSeq 1500.

How should I store my reagents?

Please refer to page 4 of the User Guide for a detailed list of reagents and storage conditions. Most reagents should be stored at -20ºC upon arrival, except for the MAGwise beads which should be stored at 4ºC and the Coding Buffer and X solution which are stored at room temperature.

How many times can I freeze/thaw reagents?

Please try to avoid freeze/thaw cycles. For a normal kit use up to 8 freeze/thaw cycles has been validated for the cDNA synthesis module and the Library Primer Mix. Sample and pool barcode reagents are not frozen under normal storage conditions.

Can I purchase individual reagents without purchasing a plexWell library preparation kit?

X-Solution, Coding Buffer (3X), and MAGwise™ Paramagnetic Beads are available as standalone products. Contact [email protected] for volume-based pricing. Inquire at [email protected] for additional reagents or for a customized kit option.

Applicable to plexWell Rapid Single Cell RNA Library Preparation Kit.

This kit offers a high-efficiency workflow for full-length transcript coverage. Download the User Guide for more FAQs and information here.

Does plexWell Rapid Single Cell RNA Library Preparation Kit provide stranded RNA-seq information?

No, this workflow does not provide stranded information. This is in alignment with most scRNA-seq workflows.

Does plexWell Rapid Single Cell Library Preparation Kit support use of UMIs?

The oligos used during cDNA synthesis do not contain UMIs. The plexWell Rapid Single Cell Library Preparation Kit provides full-length transcript coverage and utilizes transposomes to fragment the full-length cDNA prior to downstream library preparation. UMIs incorporated during cDNA generation occur at the 3’ and 5’ ends of the cDNA molecules. Since the full length cDNA is subsequently fragmented (via transposomes) during the library preparation, any UMI information will be lost for many library fragments.

Will I cover noncoding transcripts with plexWell Rapid Single Cell RNA Library Preparation Kit?

No. The plexWell Rapid Single Cell RNA Library Preparation Kit uses a priming approach that targets polyadenylated transcripts during reverse transcription which enriches for mature mRNA transcripts.

What Reagents are required for the plexWell Rapid Single Cell RNA library Preparation Kit workflow?

The KAPA Biosystems HiFi HotStart ReadyMix PCR Kit (KK2602) now distributed by Roche (7958935001) is required for both the cDNA and library preparation modules. Additionally, common laboratory reagents such as Tris-HCl, pH 8.0, 80% ethanol and nuclease-free water are used throughout the protocol. Reagents for QC of starting samples, in the case of total RNA, the cDNA and final library are also recommended.

Can I use a different polymerase for cDNA and library amplification?

KAPA HiFi HotStart ReadyMix has been validated with plexWell library preparation kits. Other DNA amplification reagents and reaction conditions might produce adequate cDNA and library yields, but may introduce bias leading to uneven coverage.

What equipment is required for the plexWell Rapid Single Cell RNA Library Preparation Kit workflow?

A thermalcycler that can hold a fully skirted low profile 96 well PCR plate (Biorad P/N HSP9601) is required for the SB module. If you do not have thermal cyclers compatible with these plates, contact [email protected] for guidance on how to amend the protocol. Additional equipment, including a cell sorter, table-top vortex; plate centrifuge; minifuge; appropriate pipettors, magnet for MAGwise bead-based purification steps, equipment for assessing cDNA quality and library size by gel electrophoresis (BioAnalyzer, TapeStation, or Fragment Analyzer, etc.) as well as for cDNA and library concentration (fluorometer or qPCR instrument), and an Illumina sequencing system are needed. A complete list can be found on page 6 of the User Guide.

My thermocycler doesn’t hold fully skirted low-profile plates. What do I do?

Set up the SB reaction as described in the User Guide. Pipette to mix. Seal the plate and centrifuge. Unseal the plate and carefully transfer the reactions to a PCR plate that fits in your thermocycler.

What labware is required for the plexWell Rapid Single Cell RNA Library Preparation Kit workflow?

During the cDNA module, different plasticware (PCR plates, strip tubes) can affect cDNA yield and length. The workflow has been validated using segmented, semi-skirted PCR plates (Thermo Fisher P/N AB0900) for the cDNA synthesis module. Additionally, evaporation resistant seals and 96-well magnet are needed for the plates and magnetic racks for the 1.5- and 2-ml DNA LoBind tubes. Most other consumables utilized in the workflow such as 1.5- and 2-mL DNA LoBind tubes, pipet tips, and PCR are tubes are common laboratory supplies. A complete list can be found on page 6 of the User Guide.

Can I use different 96-well plates for cDNA synthesis?

No. 96-well, segmented,semi-skirted plates from ThermoFisher (P/N AB0900) have beed validated for cDNA synthesis.

Which magnets are recommended for the bead cleanups?

For tube magnets, we recommend the Dynamag-2 Magnet from ThermoFisher, but other appropriate magnets may be used provided they support the elution volumes in the protocol. Bead settling times will vary depending on the strength of the magnet used. For magnets other than the DynaMag-2, visually confirm that the supernatant has cleared before proceeding. For a plate based magnet, we recommend a 96-well side magnet (such as ThermoFisher Dynamag 96 Side) or ring magnet (such as 96S Super Magnet from Alpaqua).

Applicable to plexWell 96 Library Preparation Kit.

This kit offers an assay-ready 96-well fully-skirted low-profile PCR plate and normalizes input DNA over wide input range of 3-30 ng. Download the User Guide for more FAQs and information here.

plexWell 384 and plexWell LP384 have identical components. Can these reagents be used interchangeably? How do I know which kit to order?

The plexWell LP 384 kit contains more PB reagent, Library Primer Mix, and MAGwise™ Paramagnetic Beads than the plexWell 384 kit, but the reagent compositions are the same. plexWell LP 384 reagents can be used in conjunction with either the plexWell LP 384 or plexWell 96 or 384 protocols. Key differences between the protocols are the starting sample concentration and volume, pooling volume, and default level of plexing per pool. If both the plexWell 384 and plexWell LP 384 protocols are used routinely, purchase the plexWell LP 384 kit.

Can I process <96 samples at a time with a plexWell 96 or 384 kit?

All 96-well plexWell assay-ready kits are validated using all 96 SB reagents simultaneously. For processing less than 96 samples, refer to the Appendix of the User Guide. For additional guidance contact [email protected].

If I processed <96 samples, can I reuse the remaining SB reagents?

The SB reagents can be saved if they are as-shipped (i.e. if no DNA or buffers have been added, and no incubations have been done). If you are processing <96 samples and would like to preserve the SB reagents, set the SB reaction up as described in the User Guide, but add sample and Coding Buffer only to the appropriate wells of the SB plate. Pipette to mix. Seal the SBX plate, then centrifuge. Unseal the plate and transfer the reactions to a new PCR plate. Reseal the SBX plate and store remaining SB reagents at -20ºC for subsequent use.

What is the volume of SB reagent in the SBX96 plate?

Each well of the SBX96 plate contains 4 µl of reagent.

Is more than one PB reagent (i5 index) available for plexWell 96 kits? Can I order two plexWell 96 kits and sequence the libraries in the same lane?

No. All plexWell 96 kits contain the same PB reagent (PBX007). If you need to sequence more than 96 samples in a single lane, purchase a plexWell 384 or LP 384 kit. In cases where <384 samples have to be processed, contact [email protected] to explore options for a customized kit.

The concentration of my input DNA samples is variable. Can I still use them?

Yes. For plexWell™ 96 and 384 kits, the average DNA concentration across all samples should be adjusted to 2.5 ng/µl by applying a global dilution factor (GDF). However, individual samples can have a higher or lower concentration. For example, if the average concentration across all your samples is 25 ng/µl before starting library prep, you would apply a 10-fold dilution factor to all your samples, to obtain a final average sample input concentration of 2.5 ng/µl. For optimal results, keep the concentration of all samples within a 10-fold range, such that the input falls in the range of 3 – 30 ng (0.75 – 7.5 ng/µl), with an average concentration of 2.5 ng/µl. Nevertheless, it is not necessary, nor recommended, to dilute every sample to exactly 2.5 ng/µl. To understand how to calculate and apply a global dilution factor, contact [email protected].

What are the expected library QC metrics?

Libraries generated with a plexWell 96 or 384 kit should have an average fragment length in the range of 500 – 850 bp, depending on sample type and average input, and a final concentration >25 nM.

What is the expected duplication rate for libraries generated with a plexWell 96 or 384 kit?

Duplication rate is impacted by sample complexity, library complexity, sequencing depth and sequencing length. plexWell 384 specifications were derived from paired-end (2 x 150 bp) sequencing of libraries made from 10 ng of E. coli genomic DNA. For an analysis based on 1 million paired reads (clusters), the duplication rate should be ≤10%.

Applicable to plexWell LP 384 DNA Library Preparation Kit.

This kit offers the same enhanced plexWell workflow engineered for low-pass whole genome library prep and sequencing. Download the User Guide for more FAQs and information here.

plexWell 384 and plexWell LP384 have identical components. Can these reagents be used interchangeably? How do I know which kit to order?

The plexWell LP 384 kit contains more PB reagent, Library Primer Mix, and MAGwise™ Paramagnetic Beads than the plexWell 384 kit, but the reagent compositions are the same. plexWell LP 384 reagents can be used in conjunction with either the plexWell LP 384 or plexWell 96 or 384 protocols. Key differences between the protocols are the starting sample concentration and volume, pooling volume, and default level of plexing per pool. If both the plexWell 384 and plexWell LP 384 protocols are used routinely, purchase the plexWell LP 384 kit.

Is there a 96-reaction version of the plexWell LP 384 kit?

A plexWell LP 96 kit can be provided for pilot projects, demonstrations, or through a custom kit order. Contact [email protected] to find out how to order a plexWell Low Pass 96 kit.

Can I process <96 samples at a time with the LP 384 kit?

The standard plexWell LP384 protocol is designed for pools of 48 samples, but assumes that 96 samples will be processed at once. For pools of 48 samples, set the SB reaction up as described in the User Guide, but add sample and Coding Buffer only to the appropriate wells of the SB plate. Prior to thermocycling, pulse-fuge the SBX plate. Unseal, then transfer the contents of the DNA-containing wells to a new PCR plate. Reseal and store the original SB plate at -20ºC. Place the new PCR plate in the thermocycler for the TAG reaction. For processing of pools <48, refer to the appendix in the User Guide appendix or contact [email protected].

If I processed <96 samples, can I reuse the remaining SB reagents?

The SB reagents can be saved if they are as-shipped (i.e. if no DNA or buffers have been added, and no incubations have been done). If you are processing <96 samples and would like to preserve the SB reagents, set the SB reaction up as described in the User Guide, but add sample and Coding Buffer only to the appropriate wells of the SB plate. Pipette to mix. Seal the SBX plate, then centrifuge. Unseal the plate and transfer the reactions to a new PCR plate. Reseal the SBX plate and store remaining SB reagents at -20ºC for subsequent use.

I ordered multiple plexWell LP 384 index sets. Do I need to match the SBX and PBX lot numbers?

No. All PBX reagents are compatible with all SBX reagents. It is not necessary to segregate Box 1 and Box 2 components for specific Box 3 reagents.

What is the volume of SB reagent in the SBX96 plate?

Each well of the SBX96 plate contains 4 µl of reagent.

The concentration of my input DNA samples is variable. Can I still use them?

Yes. For plexWell™ 96 and 384 kits, the average DNA concentration across all samples should be adjusted to 1.67 ng/µl by applying a global dilution factor (GDF). However, individual samples can have a higher or lower concentration. For example, if the average concentration across all your samples is 17 ng/µl before starting library prep, you would apply a 10-fold dilution factor to all your samples, to obtain a final average sample input concentration of 1.7 ng/µl. For optimal results, keep the concentration of all samples within a 5-fold range, such that the input falls in the range of 5 – 25 ng (0.83 – 4.17 ng/µl), with an average concentration of 1.67 ng/µl. Nevertheless, it is not necessary, nor recommended, to dilute every sample to exactly 1.67 ng/µl. To understand how to calculate and apply a global dilution factor, contact [email protected].

What are the expected library QC metrics?

Libraries generated with a plexWell LP 384 kit should have an average fragment length in the range of 500 – 850 bp, depending on sample type and average input, and a final concentration >20 nM.

What is the expected duplication rate for libraries prepared with a plexWell LP 384 kit?

Duplication rate is impacted by sample complexity, library complexity, sequencing depth and sequencing length. plexWell LP384 kit specifications were derived from paired-end (2 x 150 bp) sequencing (on a NovaSeq system) of libraries made from 10 ng of human genomic DNA. For an analysis based on 10 million paired reads (clusters), the duplication rate should be ≤10%. Due to the nature of patterned flow cells, the duplication rate may vary depending on loading efficiency. Customers have sequenced human samples to an average depth of 4X, with resulting duplication rates <20%.

Applicable to plexWell 384 DNA Library Preparation Kit.

This kit offers assay-ready 96-well fully-skirted low-profile PCR plates (4 x 96). Download the User Guide for more FAQs and information here.

plexWell 384 and plexWell LP384 have identical components. Can these reagents be used interchangeably? How do I know which kit to order?

The plexWell LP 384 kit contains more PB reagent, Library Primer Mix, and MAGwise™ Paramagnetic Beads than the plexWell 384 kit, but the reagent compositions are the same. plexWell LP 384 reagents can be used in conjunction with either the plexWell LP 384 or plexWell 96 or 384 protocols. Key differences between the protocols are the starting sample concentration and volume, pooling volume, and default level of plexing per pool. If both the plexWell 384 and plexWell LP 384 protocols are used routinely, purchase the plexWell LP 384 kit.

Can I process <96 samples at a time with a plexWell 96 or 384 kit?

All 96-well plexWell assay-ready kits are validated using all 96 SB reagents simultaneously. For processing less than 96 samples, refer to the Appendix of the User Guide. For additional guidance contact [email protected].

If I processed <96 samples, can I reuse the remaining SB reagents?

The SB reagents can be saved if they are as-shipped (i.e. if no DNA or buffers have been added, and no incubations have been done). If you are processing <96 samples and would like to preserve the SB reagents, set the SB reaction up as described in the User Guide, but add sample and Coding Buffer only to the appropriate wells of the SB plate. Pipette to mix. Seal the SBX plate, then centrifuge. Unseal the plate and transfer the reactions to a new PCR plate. Reseal the SBX plate and store remaining SB reagents at -20ºC for subsequent use.

What is the volume of SB reagent in the SBX96 plate?

Each well of the SBX96 plate contains 4 µl of reagent.

I ordered multiple plexWell 384 index sets. Do I need to match the SBX and PBX lot numbers?

No. All PBX reagents are compatible with all SBX reagents. It is not necessary to segregate Box 1 and Box 2 components for specific Box 3 reagents.

The concentration of my input DNA samples is variable. Can I still use them?

Yes. For plexWell™ 96 and 384 kits, the average DNA concentration across all samples should be adjusted to 2.5 ng/µl by applying a global dilution factor (GDF). However, individual samples can have a higher or lower concentration. For example, if the average concentration across all your samples is 25 ng/µl before starting library prep, you would apply a 10-fold dilution factor to all your samples, to obtain a final average sample input concentration of 2.5 ng/µl. For optimal results, keep the concentration of all samples within a 10-fold range, such that the input falls in the range of 3 – 30 ng (0.75 – 7.5 ng/µl), with an average concentration of 2.5 ng/µl. Nevertheless, it is not necessary, nor recommended, to dilute every sample to exactly 2.5 ng/µl. To understand how to calculate and apply a global dilution factor, contact [email protected].

What are the expected library QC metrics?

Libraries generated with a plexWell 96 or 384 kit should have an average fragment length in the range of 500 – 850 bp, depending on sample type and average input, and a final concentration >25 nM.

What is the expected duplication rate for libraries generated with a plexWell 96 or 384 kit?

Duplication rate is impacted by sample complexity, library complexity, sequencing depth and sequencing length. plexWell 384 specifications were derived from paired-end (2 x 150 bp) sequencing of libraries made from 10 ng of E. coli genomic DNA. For an analysis based on 1 million paired reads (clusters), the duplication rate should be ≤10%.

Applicable to plexWell Plus 24 DNA Library Preparation Kit.

This kit offers a more flexible format for different multiplex batch sizes (8 to 96). Download the User Guide for more FAQs and information here.

Is there a protocol for processing >24 samples using the plexWell Plus 24 Kit?

Yes. Up to 96 libraries can be processed in four batches of 24 samples each, or in other combinations, as described in the plexWell Plus 24 User Guide.

Are plexWell Plus 24 kits with more than four i5 indices available?

Not currently. Each plexWell Plus 24 kit is provided with the same four Pool Barcode Reagents (i5 indices). For custom configurations, contact [email protected]. Alternatively, for routine processing of >96 samples, consider using a plexWell 96 or 384 kit for batches of 96, and a plexWell Plus 24 kit for the remaining samples.

If I make multiple libraries in batches of 24 samples using a plexWell Plus 24 Kit, can I pool those libraries together in the same sequencing run?

Yes. plexWell Plus libraries prepared from up to 96 samples can be sequenced together.

What are the expected library QC metrics?

Libraries generated with a plexWell Plus 24 kit should have an average fragment length in the range of 500 – 850 bp, depending on sample type and average input; and a final concentration >25 nM for a 24-plex or >17 nM for an 8-plex.

What is the expected duplication rate for libraries prepared with a plexWell Plus 24 kit?

Duplication rate is impacted by sample complexity, library complexity, sequencing depth, and sequencing length. plexWell Plus 24 specifications were derived from paired-end (2 x 150 bp) sequencing of libraries made from 10 ng of E. coli genomic DNA. For an analysis based on 1 million paired reads (clusters), the duplication rate should be ≤10%.

Are libraries made with plexWell Plus 24 kit compatible with libraries prepared with a plexWell-96 or 384 kit? What about compatibility with libraries made with kits from other manufacturers?

Libraries made with plexWell Plus 24 kits are fully compatible with libraries made with plexWell 96 or plexWell 384 Library Preparation Kits (i.e. these libraries can be pooled and sequenced in the same run). plexWell libraries should be compatible with most other libraries prepared for Illumina® sequencing. However, if multiplexing with another type of library, double-check that there are no barcode conflicts.

Applicable to plexWell WGS 24 DNA Library Preparation Kit.

This kit offers 24-plex for higher-complexity human/plant/animal gDNA library prep. Download the user guide for more FAQs and information here.

Is the plexWell WGS 24 kit available for multiplexing >24 samples?

The standard plexWell WGS 24 kit is configured as an assay-ready kit for pools of 24 samples. Additional PB reagents are available in custom configurations to support multiplexing up to 96 samples. Inquire at [email protected].

Can I process <24 samples with the plexWell WGS 24 Kit?

The plexWell WGS 24 protocol was validated using all 24 SB reagents in a single pool. For ≥18 samples, you can follow the standard protocol, and adjust the pooling volume to account for the fewer samples (i.e. standard pool volume x 24/N, where N is the number of samples processed). For recommendations on processing fewer than 18 samples, contact [email protected].

If I processed <24 samples, can I reuse the remaining SB reagents?

The SB reagents can be saved if they are as-shipped (i.e. if no DNA or buffers have been added, and no incubations have been done). If you are processing <24 samples and would like to preserve the SB reagents, set the SB reaction up as described in the User Guide, but add sample and Coding Buffer only to the appropriate wells of the SB plate. Pipette to mix. Seal the SBW plate, then centrifuge. Unseal the plate and transfer the reactions to a new PCR plate. Reseal the SBW plate and store remaining SB reagents at -20ºC for subsequent use.

What is the volume of SB reagent in the SBW24 plate?

Each well of the SBW24 plate contains 8 µl of reagent.

Does this kit auto-normalize?

No. This kit has been optimized for 200 ng input in each well.

What is the working sample input range for this kit?

The sample input is fixed at 200 ng in an 8 µl reaction volume. If your sample concentrations are <25 ng/µl, but you have sufficient sample for a 200 ng input, contact [email protected] for suggestions on how to modify the SB, stop and purification reactions to work with your samples.

What are the expected library QC metrics?

Libraries generated with a plexWell WGS 24 kit should have an average fragment length in the range of 600 – 800 bp, depending on sample type and average input, and a final concentration >25 nM.

What is the expected duplication rate for libraries prepared with a plexWell WGS 24 kit?

Duplication rate is impacted by sample complexity, library complexity, sequencing depth and sequencing length. plexWell WGS 24 kit specifications were derived from paired-end (2 x 150 bp) sequencing (on a NovaSeq system) of libraries made from 200 ng of human genomic DNA. For an analysis based on 400 million paired reads (clusters), the duplication rate should be ≤10%.

Applicable to purePlex DNA Library Preparation Kit.

This kit offers speed, performance, and auto-normalization with unique dual indexes. Download the User Guide for more FAQs and information here.

What applications are recommended for the purePlex Library Preparation Kit?

The purePlex Library Preparation Kit is recommended for synthetic construct sequencing (amplicons, plasmids, etc.), low-pass whole genome sequencing, whole small genome sequencing (<50 Mb), single cell RNA-seq (scRNA-seq), and metagenomics/microbiome sequencing.

How many samples can be processed with a single kit?

A single purePlex kit provides enough reagents to process up to 96 samples.

Are all required adapters, indices, and amplification primers included in purePlex Library Preparation Kit?

Yes. The purePlex kit includes all the indexed adapters and amplification primers necessary to make dual-indexed Illumina-compatible libraries. It is worth noting that the i7 index is added by the i7-Tagging Reagent (i7-TR) and the i5 index is added by the i5-Tagging Reagent (i5-TR).

Are any additional reagents, consumables, or equipment needed?

Reagents: KAPA HiFi HotStart ReadyMix; 10 mM Tris-HCl, pH 8.0, ultra-pure water, ethanol, reagents for DNA and library QC (PicoGreen), and Illumina sequencing kits. Consumables: 1.5 mL LoBind tubes; PCR plate, PCR strip tubes or individual tubes; pipette tips; plate seals or strip caps. Equipment: Table-top vortex; plate centrifuge; minifuge; appropriate pipettors, magnet (suitable for 1.5 ml LoBind tube) for MAGwise bead-based purification steps; a thermal cycler, equipment for assessing library size by gel electrophoresis (BioAnalyzer, TapeStation, or Fragment Analyzer, etc.) and library concentration (fluorometer or qPCR instrument), and an Illumina sequencing system.

Are MAGwise purification beads the same as other purification beads?

MAGwise purification beads are similar to many other commercially available purification beads. The purePlex kit has been validated using MAGwise purification beads, thus it is recommended to use the included MAGwise purification beads for optimal performance. However, other SPRI purification beads should perform similarly and can be utilized/implemented with optimization.

The MAGwise purification beads were accidentally stored at the wrong temperature. Can it still be used?

To better understand the potential impact and for guidance, contact [email protected]. Short term storage of MAGwise at room temperature (<2 weeks) or -20°C (<2 days) does not appreciably alter performance for most applications.

Coding buffer and X-solution were accidentally stored at the wrong temperature. Can they still be used?

Yes. Equilibrate the coding buffer and X-solution to room temperature by removing them from the refrigerator and/or defrosting them. Before use, ensure they are homogenous. Coding Buffer should be vortexed then pulse-fuged. X-solution should be checked for precipitate. If precipitate is present in the X-solution, warm to 37°C, then invert or pipet to mix. Do NOT vortex X-Solution. Store remaining Coding Buffer and X-Solution at room temperature.

Is there flexibility in pooling for the purePlex Library Preparation Kit?

Yes. The purePlex Library Preparation Kit workflow has been optimized and validated for 24 samples per pool, with columns 1-3 in pool A, columns 4-6 in pool B, columns 7-9 in pool C and columns 10-12 in pool D. However, because both the i7 and i5 tagging steps are performed on individual samples, the purePlex kit allows flexibility in pooling and batch sizes. Please see the User Guide for alternate pooling strategies and recommendations for best practices.

The concentration of DNA sample input is variable. Can the samples still be prepped together?

The purePlex Library Preparation Kit performs optimally with 25 ng of dsDNA per well, however, individually adjusting each sample to 5 ng/µl is not necessary as purePlex Library Preparation Kits are formulated to tolerate up to a 10-fold difference in sample input (5 to 50 ng).

Can a different index combination of i7-Tagging Reagent and i5-Tagging Reagent for the purePlex Library Preparation Kit be used?

No. The purePlex Library Preparation Kit has been optimized for the specific combination of i7-TR and i5-TR. Alternative combinations may affect autonormalization, fragment length, library concentration and complexity. However, incorrect combinations of i7-i5 tagging reagent are likely to generate sequence-able library and depending on the sequencing application, it is possible to move forward with the library preparation and sequencing without impacting the sequencing results.

Applicable to ExpressPlex Library Preparation Kit.

This kit offers a 90-minute library prep (for 96 samples), minimal liquid handling steps, and one pool cleanup. Download the FAQs document and User Guide for more information here and here, respectively.

What applications are recommended for the ExpressPlex Library Preparation Kit?

The ExpressPlex library preparation kit is recommended for synthetic construct sequencing (amplicons, plasmids, etc.).

Are all required adapters, indices, amplification master mix and amplification primers included in ExpressPlex library preparation kit?

Yes. The ExpressPlex kit includes all the indexed adapters, amplification master mix, and amplification primers necessary to make dual-indexed Illumina-compatible libraries.

How many samples can I batch together?

The ExpressPlex kit allows flexible batching. Each assay-ready plate can process up to 96 samples. Up to 384 samples can be processed per kit.

If I processed <96 samples, can I reuse the remaining reagents?

The Ready Reaction reagents can be saved if they are as-shipped (i.e. if no DNA or Indexing reagent have been added, and no incubations have been done). If processing <96 samples and would like to preserve the Ready Reaction reagents, set the reaction up as described in the User Guide, but add sample and Indexing reagent only to the appropriate wells of the Ready Reaction plate. Pipette to mix. Seal the Ready Reaction plate, then centrifuge. Unseal the plate and transfer the reactions to a new PCR plate. Reseal the Ready Reaction plate and store remaining SB reagents at -20ºC for subsequent use.

How many total combinations are commercially available?

seqWell offers a total of 1536 index combinations in the ExpressPlex Library Prep Kit. If more than 1536 index combinations are required, please contact [email protected].

What is the recommended DNA input range for the ExpressPlex Kit?

The recommended input range for the ExpressPlex Kit is 8 - 40 ng (2 - 10 ng/µl). The kit uses 4 µl of purified DNA sample. Use of less than 8 ng of samples is not recommended due to increased risk of failure.

The concentration of DNA sample input is variable. Can the samples still be prepped together?

The ExpressPlex library preparation kit performs optimally with 16 ng of dsDNA per reaction, however, individually normalizing each sample to 4 ng/µl is not necessary as ExpressPlex library preparation kits are formulated to tolerate up to a 5-fold difference in sample input (8 to 40 ng).

What size range of amplicons is suitable for making ExpressPlex libraries?

ExpressPlex library prep is recommended for PCR products >350 bp in length. The efficiency of amplicon tagging is lower for shorter amplicons and within 50 bp of the amplicon termini, so PCR primers should be designed to prime at least 50 bases upstream from the region of interest.

What quantification methods are recommended for plasmids and PCR products?

The ExpressPlex Kit is sensitive to dsDNA concentration outside the recommended range. Fluorometric methods for dsDNA (e.g., PicoGreen, Qubit) are generally more reliable for assaying miniprepped plasmids than spectrophotometric methods. Regardless of the quantification methods employed, the purity of the DNA should be considered. There are several contaminants of plasmids that interfere with quantification including protein, genomic DNA, ssDNA and RNA. The presence of these contaminants inflates the apparent DNA concentration.

I see untagged amplicons or plasmids in my ExpressPlex purified library. Does this affect my library quantification?

qPCR based library quantification will be unaffected. Methods that quantify total DNA content (e.g., PicoGreen, Qubit) will require an adjustment. Use the TapeStation, Fragment Analyzer, or similar equipment to conduct a region analysis in determine the percentage of the DNA mass that is library (see User Guide for more details), then multiply this percentage of your DNA in the clusterable range to determine the library content.

I see untagged amplicons or plasmids in my ExpressPlex purified library. Will unfragmented amplicons or plasmids in purified libraries interfere with sequencing?

Untagged amplicons or plasmids do not interfere with clustering on the flowcell or the data sequencing quality. However, it may affect library quantification and thus the optimal loading density (see User Guide to properly adjust library quantification).

What is the expected fragment size of the ExpressPlex library? Can fragments shorter / longer than this size be sequenced?

The expected fragment size of the ExpressPlex library is 400 – 1,000 bp. If amplicons shorter than 1000 bp are used as input into ExpressPlex reactions, the resulting library fragment size distribution will be shorter than the input amplicons.

What is the compatibility of ExpressPlex 384-plex / 1536-plex libraries?

There are 4 different i5 index sets per ExpressPlex library prep kit (384 Reactions). Sets 1000 – 4000 are designed so their i5 base composition is color-matched and compatible within each set. Sets 1000 – 4000 can be sequenced together for multiplexing up to 1,536 in a single sequencing run.