FAQs

plexWell™ Library Prep Kits FAQs:

Are all required adapters, indices, and amplification primers included in plexWell Plus 24, plexWell 96, and plexWell 384 library preparation kits?

Yes. All plexWell kits include all the indexed adapters and amplification primers necessary to make dual-indexed Illumina-compatible libraries. It is worth noting that the Sample Barcode (i7 index) is added by the Sample Barcode Reagent, and the Pool Barcode (i5) is added by the Pool Barcode Reagent.

Are any additional reagents, consumables, or equipment needed?

Reagents: KAPA HiFi HotStart ReadyMix; 10 mM Tris-HCl, pH 8.0, ultra-pure water, reagents for DNA quantification (PicoGreen), and Illumina sequencing kits.

Consumables: 1.5 mL LoBind tubes; PCR strip tubes and individual tubes; pipette tips; plate seals or strip caps.

Equipment: Table-top vortex; plate centrifuge; minifuge; appropriate pipettors, magnet for MAGwise bead-based purification steps; a thermal cycler compatible with low profile fully-skirted Bio-Rad hard-shell PCR plates; 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.

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

Yes.  The average DNA concentration across the samples should be adjusted to 2.5 ng/µl by applying a global dilution factor to all the samples, but individual samples can be higher or lower.  For example, if the average concentration across all your samples is 25 ng/µl before starting library prep, you would apply a ten-fold dilution factor to all your samples, so the final average sample input concentration is 2.5 ng/µl.  You will get the best results when you keep the concentration of all samples within a 10-fold input range (0.75 – 7.5 ng/µl), but it is not necessary nor recommended to dilute every sample to the same concentration.

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.  Alternative polymerases and amplification conditions might produce adequate yields of library, but could do so at the risk of uneven coverage.

The size of my library insert looks long on the Bioanalyzer/gel/TapeStation/Fragment Analyzer.  Will it still sequence?

plexWell libraries sometimes produce longer fragments, especially as compared to library prep methods that employ mechanical shearing.  Generally speaking, longer fragments (>1000 bp) make minor contributions to library quantification and do NOT cluster efficiently on sequencers, thus they do not contribute to sequencing data.  Provided that the majority of library fragments are shorter than 1000 bp, longer library fragments are not generally a concern.

How long are the plexWell adapters?

The combined length of adapters and indexes are 135 bp.

What is the purpose of the i5 index if it is not always required for sequencing?

Multiple plexWell libraries with the same Sample Barcode (i7 index) can be pooled and sequenced together, IF they have different Pool Barcodes (i5 indices).

Do I need special sequencing primers or sequencing reagents?

plexWell libraries are sequenced using the same primers as Nextera^® libraries.  plexWell libraries are compatible with the MiSeq, NextSeq 500, HiSeq and NovaSeq sequencing systems. However, 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, GAIIx, and HiScanSQ.

Do I need to sequence with dual-indexing if I only used one PB Reagent (i5 index) on the run?

It is generally recommended to run in dual-indexing mode.  However, if a plexWell library with only one i5 index is loaded on the sequencing run, you can demultiplex based solely on the Sample Barcode (i7 index) and run in single-indexing mode. In this case, ensure that the i7 index is read.

Do you provide sample sheet templates for the MiSeq, and other systems?

Yes. Please see the links to sample sheet templates on the seqWell website.

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

Yes. Up to 96 libraries can be processed in four batches of 24 samples, or in other combinations, as described in the kit user guide.

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

Yes. plexWell Plus libraries prepared from up to 96 samples can be loaded on the same sequencing run.

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

Not currently. There are only four Pool Barcode Reagents (i5 indices) provided with each standard plexWell Plus 24 Library Prep Kit. Additional Pool Barcode Reagents may be available separately, however. Please inquire.

What is the compatibility of libraries made with plexWell Plus 24 kits with libraries made with the plexWell-96 or -384 kits? 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 loaded on to the same sequencer run). plexWell libraries should also be compatible with most other dual-indexed Nextera-style adapter libraries, but the i5 indices must be double-checked to verify they are different in order to avoid potential barcode collisions.

My samples are all more concentrated than 2.5 ng/µl.  Can I use them as is?

Not yet. First apply a single global dilution factor to all samples to bring the average concentration across all samples to 2.5 ng/µl as described above and in the kit user guide.  The SB Reagents and PB Reagents supplied in standard plexWell kits are optimized for an average input of 10 ng dsDNA input per well (4 µl of 2.5 ng/µl DNA input). If the average concentration is much greater than 2.5 ng/µl, the resulting library fragment distribution could be broader and longer than usual.

My amplicons are shorter than 1 kb.  Can I still use plexWell library preparation?

Yes.  The protocol can be used for amplicons as short as 400-500 bp, although there will be some impact on yield and insert size when compared to libraries prepared from amplicons of >1000 bp. Consider optimizing the purification conditions following library amplification (e.g., use 0.85 – 1 volume equivalents of MAGwise to recover shorter library fragments).  Please note that the depth of coverage near the termini of PCR products will be lower than in other regions. PCR primers should be designed that hybridize 75 – 100 bases upstream and downstream of the region of interest to ensure adequate depth of coverage.

Is plexWell supported for sequencing human DNA?

Yes. All plexWell library prep kits will produce high quality libraries from human genomic DNA.  However, for generating libraries with sufficient diversity and uniformity for human whole genome sequencing (WGS) at higher depths of coverage, we specifically recommend the use of plexWell WGS-24 Library Preparation Kits (seqWell P/N: WGS24).

Are plexWell libraries compatible with downstream target capture?

The use of plexWell libraries with target capture has not been tested extensively.  plexWell libraries have full-length Illumina, Nextera-style adapters with 8 base i5 and i7 indices.  As such, they should be compatible with downstream target capture when combined with appropriate (Nextera-style) adapter and index blockers during hybridization.