- Product line overview
- Technical notes
- Featured kits
- Technology and application overviews
- FAQs and tips
- DNA-seq protocols
- Bioinformatics resources
When starting with DNA of limited quantity or varying quality, the library preparation method must be accurate and efficient, avoiding purification steps that reduce sample recovery. Our line of DNA-seq products were developed to meet those needs, but we're sure you have questions about which one is best to maximize the results for your precious sample.
The following FAQ covers general questions about several of our DNA-seq kits, but is not intended to be fully inclusive. If you have any additional questions not covered by the information below, please feel free to reach out to ask one of our PhD-level sales team or technical support scientists.
General questions about our DNA-seq kits
What are the storage conditions for your DNA-seq kits?
In general, our DNA-seq kits should be stored at –20°C. Consult your product's Certificate of Analysis for specific storage guidelines.
What is the shelf life of your DNA-seq kits?
In general, the shelf life of our DNA-seq kits is one year from date of receipt under proper storage conditions. Consult your product's Certificate of Analysis for specific shelf life information.
Is a high-fidelity enzyme used in the amplification reaction?
Yes, a high-fidelity, high-processivity, low-bias DNA polymerase is used in our DNA-seq kits.
Can the DNA-seq kits be run on any thermal cycler?
Our DNA-seq kits require the use of a thermal cycler that can accommodate a 50-µl final reaction volume. Please consult the manual of your PCR instrument.
What reagents not supplied are necessary to generate sequencing-ready libraries using your DNA-seq kits?
Following generation of libraries using our DNA-seq kits, the samples will need to be purified using Agencourt AMPure XP beads (Beckman Coulter, Cat. No. A63880). Consult your product’s user manual for a complete list of required materials.
Can all of the reagents in your DNA-seq kits be mixed by vortexing?
No, only buffers and nuclease-free water may be vortexed, and should then be spun down briefly prior to use. Enzymes should be mixed by gentle pipetting and then spun down briefly prior to use. Index Plates should be spun down briefly prior to use.
Can your DNA-seq kits be used for methylation studies?
No, our DNA-seq kits have not been optimized for methylation analyses. Instead, we recommend our EpiXplore Meth-Seq DNA Enrichment Kit.
Can libraries created using your DNA-seq kits be used for microarray or PCR analyses?
Yes, the libraries created using our DNA-seq kits can be used for microarray and PCR analyses.
What is the recommended fluorescent dye for real-time PCR monitoring?
EvaGreen fluorescent dye (EvaGreen Dye, 20X in water; Biotium Cat. No. 31000) is recommended because of its high sensitivity and low interference with the amplification chemistry of our DNA-seq kits.
Can a library be prepared without any PCR amplification?
PCR amplification is required for libraries prepared using our DNA-seq kits. Please refer to the user manual for your kit to obtain detailed information about library amplification step(s).
How long, and at what temperature, should amplified libraries be stored at before additional amplification?
For best performance, unpurified, amplified libraries prepared using our DNA-seq kits should be kept at –20°C for no more than seven days before additional amplification and/or purification. Please refer to the user manual for your kit for additional instructions.
Do the DNA-seq kits use single or dual indexes?
There are a variety of different indexing options available for use with our DNA-seq kits. Choose from the following single and dual index kits:
DNA HT Dual Index kits
DNA HT Dual Index kits are designed for use with ThruPLEX and PicoPLEX library preparation kits to construct libraries for multiplexed sequencing on Illumina sequencers. These kits contain indexed PCR primers carrying the Illumina Nextera® XT v2 index sequences and offer a total of 384 dual indexes for multiplexing of up to 384 samples. The indexed PCR primers are supplied pre-dispensed in four different barcoded index plates, each of which contains one-fourth of the possible dual index combinations (Cat. Nos. R400660–R400663). They are alternately available in a set of 24 individual tubes, each containing a different dual index combination (Cat. No. R400664). Each dual index tube (24N) contains sufficient volume for up to two uses. Each well of a dual index plate (96N Sets A–D) contains sufficient volume for a single use.
DNA Unique Dual Index kits
DNA Unique Dual Index kits are designed for use with ThruPLEX and PicoPLEX library preparation kits to construct libraries for multiplexed sequencing on Illumina sequencers. These kits contain indexed PCR primers carrying the "IDT for Illumina UD" index sequences and offer a total of 96 dual indexes for multiplexing up to 96 samples. The indexed PCR primers are supplied pre-dispensed in four different sets of 24 individual tubes, each of which contains one-fourth of the possible dual index combinations (Cat. Nos. R400665–R400668). Each dual index tube (24U Sets A–D) contains sufficient volume for up to two uses.
DNA Single Index kits
DNA Single Index kits are designed for use with ThruPLEX and PicoPLEX library preparation kits to construct libraries for multiplexed sequencing on Illumina sequencers. These kits contain indexed PCR primers carrying the "TruSeq LT set A" index sequences and offer a total of 12 single indexes for multiplexing of up to 12 samples. The indexed PCR primers are supplied pre-dispensed in four different sets of 12 individual tubes, each containing a different index sequence (Cat. Nos. R400695 and R400697). Each single index tube (12S Sets A–B) contains sufficient volume for up to eight uses.
Questions about specific DNA-seq kits
Questions about the PicoPLEX Gold Single-Cell Kit
What applications are recommended for the PicoPLEX Gold Single Cell DNA-Seq Kit?
The PicoPLEX Gold Single Cell DNA-Seq Kit is used to amplify genomic DNA from single cells to reliably detect chromosomal aneuploidies, copy number variations (CNVs), and single nucleotide variants (CNVs) using next-generation sequencing on Illumina platforms.
Applications include preimplantation genetic testing (PGT) using blastomeres and trophectoderm cells as well as analysis of chromosomal abnormalities and mutations in circulating tumor cells for cancer research.
Can I use the PicoPLEX Gold Single Cell DNA-Seq Kit for whole-genome de novo sequencing from a single cell?
No. The PicoPLEX Gold Single Cell DNA-Seq Kit offers robust and reproducible amplification of DNA from single cells for the detection of copy number variations (CNVs), single nucleotide variants (SNVs), and other chromosomal anomalies. This kit should not be used for high-coverage deep sequencing such as de novo sequencing and/or complete (whole genome) resequencing.
How rapid is the PicoPLEX Gold Single Cell DNA-Seq Kit?
The PicoPLEX Gold Single Cell DNA-Seq Kit lyses cells and prepares appropriately-barcoded NGS-ready libraries in less than 3 hours.
What cell types have been successfully used with the PicoPLEX Gold Single Cell DNA-Seq Kit?
Single blastomeres, trophectoderm cells, and cultured cells have been used successfully.
Can cells stained with surface antibodies be used as input?
Yes, cells stained with surface antibodies can be used if the cells are not fixed.
Do we have to purchase barcoded oligonucleotides separately?
Our DNA single index, unique dual index, and HT dual index kits are compatible with the PicoPLEX Gold Single Cell DNA-Seq Kit.
Can we use barcodes designed in our laboratory?
No. Only DNA single index, unique dual index, and HT dual index kits have been approved for use with the PicoPLEX Gold Single Cell DNA-Seq Kit.
What are the indexing options for the PicoPLEX Gold Single Cell DNA-Seq Kit?
The DNA single index, unique dual index, and HT dual index kits are compatible with the PicoPLEX Gold Single Cell DNA-Seq Kit.
How many bases should I trim for analysis of the sequencing reads?
The first 11 cycles of each read will contain quasi-random bases introduced during the PicoPLEX Gold Single Cell DNA-Seq library preparation. For sequence alignment, either trim the initial 14 bases from each read or begin calibration and data collection at base position 15.
Can the PicoPLEX Gold Single Cell DNA-Seq Kit be used with Ion Torrent or Pac Bio platforms?
No, this kit is not compatible with Ion Torrent or Pac Bio.
Can I prepare samples for both single- and paired-end NGS sequencing?
Libraries prepared using the PicoPLEX Gold Single Cell DNA-Seq Kit are compatible with both single- and paired-end sequencing on Illumina platforms.
What is the size range of fragments expected with libraries prepared using the PicoPLEX Gold Single Cell DNA-Seq Kit?
Libraries prepared using the PicoPLEX Gold Single Cell DNA-Seq Kit result in a broad size-range distribution of fragments, typically ranging from ~300–1,000 bp total size (~200–900 bp insert size).
Can the PicoPLEX Gold Single Cell DNA-Seq Kit be run on any thermal cycler?
The PicoPLEX Gold Single Cell DNA-Seq Kit must be run on a thermal cycler that can accommodate 50-µl reaction volumes and that is equipped with a heated lid. Set the temperature of the heated lid to 100–105°C to avoid sample evaporation during incubation and cycling.
Can the PicoPLEX Gold Single Cell DNA-Seq Kit be used without a real-time PCR machine?
A real-time PCR cycler is recommended to monitor amplification by the addition of fluorescent dye (not provided with the kit) to the reaction. If a regular thermal cycler is used instead, there is no need to add the dye. Substitute with an appropriate amount of nuclease-free water to adjust volume while preparing the Amplification Reaction Master Mix. In the absence of a real-time thermal cycler, use a regular thermal cycler and quantify library amplification via gel or Bioanalyzer analysis.
What is the recommended fluorescent dye to monitor amplification using a real-time PCR cycler?
EvaGreen dye (Cat. No. 31000-T, Biotium Inc.) is recommended due to its high sensitivity and low interference with amplification chemistry. Depending on the real-time instrument used, a calibration dye may also be needed. Please refer to your real-time PCR instrument's user manual for additional information.
How do I quantify the pooled, purified library for loading onto an Illumina sequencer?
Quantify PicoPLEX Gold Single Cell DNA-Seq libraries using real-time qPCR using the appropriate instrument-specific Illumina library quantification kit. Pooled, purified libraries are diluted 50,000–500,000-fold and used as the template for quantification by real-time qPCR. It is recommended to use 300 bp as the size for calculating the library concentration.
What concentration of PicoPLEX Gold Single Cell DNA-Seq library should be loaded onto the Illumina MiSeq flow cell?
With libraries made from a single cell using the PicoPLEX Gold Single Cell DNA-Seq Kit, a good starting point is to consider a library size of 300 bp and load 16 pM on the MiSeq® v3. Other platforms will have different loading concentrations, so we recommend following the manufacturers' instructions. It is very important to add at least 5% PhiX DNA to the library prior to loading on the flow cell to achieve optimal diversity.
What ratio of AMPure XP beads should I use for purifying the library prepared with the PicoPLEX Gold Single Cell DNA-Seq Kit?
Mix the beads and the sample(s) at a 1:1 ratio for most NGS-based CNV detection purposes.
Does the PicoPLEX Gold Single Cell DNA-Seq Kit's coverage span GC-rich regions?
More than 90% of the reads from libraries generated with the PicoPLEX Gold Single Cell DNA-Seq Kit fall between 25% and 70% GC content.
Questions about the PicoPLEX Single-Cell WGA Kit
What are the strengths of PicoPLEX versus other WGA technologies?
The greatest advantage of PicoPLEX over other WGA technologies is the reproducibility obtained from cell to cell while using single cells. This technology also yields a very low background.
What are the recommended DNA inputs for PicoPLEX NGS kits?
- 1–10 human cells (e.g., blastomeres, polar bodies, trophoblasts, amniocytes, CTCs, or cultured cells)
- 1,000–10,000 bacterial cells
- Intact or fragmented, single- or double-stranded DNA
- Isolated DNA (15–60 pg of human DNA)
NOTE: Inputs higher than 60 pg are possible, but require cycle optimization at certain steps
- Input size over 500 bases
- Sorted chromosomes
What cell types have been successfully amplified by the PicoPLEX Single Cell WGA Kit?
Single blastomeres, polar bodies, trophoblasts, amniocytes, and cultured cells have been amplified successfully.
Should cells be washed before collection?
Yes. Cells should be washed to minimize extracellular DNA or growth media contaminants. We recommend washing in PBS (Ca2+-free, Mg2+-free, and BSA-free) and limiting carryover of wash buffer to less than 2.5 μl.
Is there a nonstick wash buffer for washing single cells prior to using PicoPLEX NGS kits?
While this has not been validated at Takara Bio, some customers have reported successfully using polyvinyl alcohol (0.1% PVA, for example) in PBS to wash/collect their cells. Additionally, your washing/collecting buffer (PBS) must be Ca2+- and Mg2+-free.
What cell collection methods are compatible with PicoPLEX NGS kits?
Flow sorting, dilution, and micromanipulation are compatible with PicoPLEX NGS kits.
Are there special requirements for flow sorting?
We strongly recommend using light scattering or phase contrast, and not fixing, to sort or collect samples. Microscopic/visual confirmation of successfully sorted cells can be used to optimize sorting conditions.
What is the sample input volume for the PicoPLEX Single Cell WGA Kit?
The kit will accommodate a 5-μl sample input volume per reaction, with a maximum of 2.5 μl of cell and buffer carryover.
Sigma-Aldrich sells a GenomePlex WGA kit. Does it contain the same technology as the PicoPLEX Single Cell WGA Kit?
No, the PicoPLEX Single Cell WGA Kit uses different technology.
How rapid is the PicoPLEX Single Cell WGA Kit?
The PicoPLEX Single Cell WGA Kit lyses cells and amplifies to >5 μg of end product DNA in <3 hours.
How robust is the PicoPLEX Single Cell WGA Kit process?
The PicoPLEX Single Cell WGA Kit has about a 90% amplification success rate with flow-sorted tissue culture cells, limited by the uncertainties of sorting rather than by amplification. Single cells always give a high, reproducible yield of amplified genomic DNA. Buffer controls should always give virtually no background.
Does the PicoPLEX Single Cell WGA Kit reproducibly amplify genomic loci?
Yes, with about a 90% correlation coefficient for qPCR Ct data from replicate single-cell reactions.
What is the expected genomic locus representation and Loss of Heterozygosity (LOH) for the product of the PicoPLEX Single Cell WGA Kit?
See the table below for third-party data obtained using the PicoPLEX Single Cell WGA Kit.
|SNP genotyping method||Single-cell amplification success rate||SNP call rates||LOH|
|Illumina Infinium SNP array||95%||50–60%||7–12%|
Can buffer control reactions be distinguished from single-cell reactions?
Yes. The PicoPLEX Single Cell WGA Kit amplifies with single-copy sensitivity and high specificity.
Is the product of this technology single- or double-stranded?
The end product of the PicoPLEX Single Cell WGA Kit is a mixture of single- and double-stranded DNA.
What NGS application(s) can the PicoPLEX Single Cell WGA Kit be used for? Which applications are specifically recommended for this kit?
The PicoPLEX Single Cell WGA Kit can be used for:
- Copy number variation (CNV) analysis: superior for oligonucleotide and array CGH
- SNP genotyping
- Mutation detection by PCR
The PicoPLEX Single Cell WGA Kit particularly excels at copy number variation analysis.
What makes the PicoPLEX Single Cell WGA Kit suited for SNP genotyping?
The PicoPLEX Single Cell WGA Kit is well suited for SNP genotyping because the same loci are reproducibly amplified between different cells. SNPs contained within well-represented regions can be accurately detected without the stochastic dropouts and noise associated with other WGA methods.
Can the PicoPLEX Single Cell WGA Kit be used for gel-based STR genotyping?
The PicoPLEX Single Cell WGA Kit has not been optimized for gel-based STR genotyping.
Can the PicoPLEX Single Cell WGA Kit be used for probe-based qPCR assays and sequencing assays?
Yes, as long as the amplimers are less than 250 bp.
How can I label PicoPLEX Single Cell WGA Kit products for microarrays?
Random-prime labeling and chemical labeling.
How much amplified DNA from the PicoPLEX Single Cell WGA Kit do I need for array and qPCR assays?
For microarrays, we recommend starting with the amount recommended in the instructions from the assay manufacturer and titrating if necessary to improve results even further.
For PCR assays, we recommend using 5–200 ng of amplified PicoPLEX Single Cell WGA Kit library.
Can the product of the PicoPLEX Single Cell WGA Kit be taken straight to NGS?
No. The PicoPLEX Single Cell WGA Kit does not have adapters nor barcodes. If NGS is the desired application, the WGA material can be used in a library preparation technology (e.g., the ThruPLEX DNA-Seq Kit).
Can I make the product of the PicoPLEX Single Cell WGA Kit NGS-ready?
Takara Bio does not support this application, but please check the following references for recommended DNA fragmentation sizes:
|Yang et al. NGS analysis of PicoPLEX WGA-amplified single-copy sorted chromosomes to infer the haplotype of individual human chromosomes. PNAS 108, 12–17 (2011).||Detailed protocol provided in the methods section.|
|Leung et al. NGS analysis of single bacteria cells using PicoPLEX WGA amplification on microfluidic device. PNAS 109, 7665–7670 (2012).||Results provided with no method revealed.|
Does Takara Bio have an NGS-ready kit for single-cell applications?
Yes. PicoPLEX Gold DNA-Seq Kits can be used with Illumina NGS platforms.
Questions about the ThruPLEX DNA-Seq Kit
What is the difference between the ThruPLEX DNA-Seq Kit and other commercially available library preparation kits?
The ThruPLEX DNA-Seq Kit is the fastest and most sensitive library prep kit available. Repair, ligation, and amplification are performed in a single tube in three simple steps.
- Input amount: 50 pg to 50 ng
- No cleanup steps
- Number of steps to library: 3
- Time to library: less than 2 hours
- Number of sample transfers: 0
Does the ThruPLEX DNA-Seq Kit’s coverage span GC-rich regions?
Yes, the ThruPLEX DNA-Seq Kit produces uniform coverage in GC-rich regions.
Can denatured/single-stranded DNA be used with the ThruPLEX DNA-Seq Kit?
No, DNA must be double-stranded for use with the ThruPLEX DNA-Seq Kit.
Is DNA fragmentation necessary?
Yes. If the DNA is >1 kb in size, it must be fragmented prior to use with the ThruPLEX DNA-Seq Kit.
What are the options available for DNA fragmentation?
DNA fragmentation can be accomplished either mechanically (e.g., Covaris, Bioruptor) or enzymatically (e.g., our DNA Fragmentation Kit).
What total amount of DNA is needed for Covaris shearing, and what type of buffer volumes are used during the shearing?
Successful shearing has been accomplished with 2–50 ng of DNA in 50–130 µl of TE or low-TE buffer. If you are working with smaller amounts of DNA, see the "Low-volume DNA shearing for ThruPLEX library prep" tech note.
What Covaris shearing specifications should be used to fragment the DNA prior to ThruPLEX DNA-Seq Kit usage?
Covaris-recommended settings should be used to obtain the desired template fragment size. Please refer to the Covaris website for additional guidance.
Where can I find information about a kit for preparing randomly fragmented genomic DNA for use with the ThruPLEX DNA-Seq Kit?
Our DNA Fragmentation Kit can be used to prepare randomly fragmented genomic DNA for use with the ThruPLEX DNA-Seq Kit by following the instructions in the DNA Fragmentation Kit User Manual.
What is the recommended DNA input range for the ThruPLEX DNA-Seq Kit?
The ThruPLEX DNA-Seq Kit has been designed to amplify an input range of 50 pg to 50 ng. The input amount depends on the desired level of complexity needed and the application.
Is it necessary to use indexing reagents? If so, which indexes should be used together for multiplexing?
Yes, it necessary to use indexing reagents, which must be purchased separately. They are supplied in tubes or a microplate and consist of amplification primers containing Illumina-compatible indexes, which must be used in the ThruPLEX DNA-Seq Library Amplification Reaction.
What is the recommended method to determine the optimal number of PCR cycles needed in the library amplification reaction?
Please refer to the ThruPLEX DNA-Seq Kit User Manual for information on selecting the optimal number of cycles for library amplification.
Can the ThruPLEX DNA-Seq Kit be used without a real-time PCR machine? If so, how do I know when to stop my run?
Yes, the ThruPLEX DNA-Seq Kit can be used even if a lab does not have access to a real-time PCR machine. Please refer to the ThruPLEX DNA-Seq Kit User Manual, which contains a table that provides a guide for selecting the number of PCR cycles based on the DNA input amount used.
Can I perform additional amplification of libraries prepared with the ThruPLEX DNA-Seq Kit if the concentrations or yields of the libraries are insufficient?
Yes, ThruPLEX DNA-Seq libraries can be further amplified without adding extra reagents after storage (4°C for up to 6 hours, or –20°C for up to 7 days). Please refer to the ThruPLEX DNA-Seq Kit User Manual for the library processing workflow and instructions on performing additional amplification cycles.
Can I perform additional amplification of ThruPLEX DNA-Seq libraries after they are purified with AMPure XP beads?
What is the expected yield for the amplified library from the ThruPLEX DNA-Seq Kit?
The amount of amplified library can range from 100 ng to 1 µg depending upon many variables, including sample type, fragmentation size, and thermal cycler used. When starting with Covaris-fragmented reference DNA with an average size of 200 bp (using the recommended number of amplification cycles in the ThruPLEX DNA-Seq Kit User Manual), typical yields range from 300 to 700 ng.
When should library quantification be performed?
Quantification must be performed prior to sequencing. It is normally done after the AMPure XP purification step, but libraries can also be quantified immediately following the Library Amplification Reaction to normalize samples prior to pooling. If yield is of concern, the quantification method used must not require purification of the library. Please refer to the ThruPLEX DNA-Seq Kit User Manual for the library processing workflow after library preparation and for instructions on library quantification.
Is it necessary to perform quantification of ThruPLEX DNA-Seq libraries before and after the AMPure XP cleanup step?
No, it is only necessary to quantify your individual or pooled libraries prior to sequencing. If you choose to quantify your libraries after the Library Amplification Reaction, it is recommended to do a final quantification of the purified libraries prior to sequencing because AMPure XP purification can result in loss of DNA.
Are there any special considerations that must be taken into account when performing AMPure XP purification on libraries prepared with the ThruPLEX DNA-Seq Kit?
Yes, we suggest using a 1:1 bead to sample ratio. Additionally, a freshly prepared solution of 80% must be used in all washing steps of the protocol. Please refer to the ThruPLEX DNA-Seq Kit User Manual for detailed instructions on AMPure XP purification of ThruPLEX DNA-Seq libraries for next-generation sequencing.
Which Illumina NGS systems can I use to sequence the libraries prepared using the ThruPLEX DNA-Seq Kit?
Libraries prepared with the ThruPLEX DNA-Seq Kit are compatible with all Illumina sequencing platforms, including the HiSeq®, MiSeq®, NextSeq® 500, GAIIx™, NovaSeq™, and MiniSeq™.
What concentration of ThruPLEX DNA-Seq library should be loaded onto a flow cell?
Please follow Illumina’s recommendations for the optimal loading concentration specific to the version of flow cell you are using. For sequencing on the Illumina MiSeq®, v3, we suggest that you load 14–15 pM of ThruPLEX DNA-Seq library.
Are there any special steps that need to be followed when loading a ThruPLEX DNA-Seq library onto a flow cell?
No, there are no special steps. ThruPLEX DNA-Seq libraries should be handled and processed in the same way as libraries generated using Illumina library preparation kits, such as the TruSeq® Nano DNA HT Sample Prep Kit.
Is it recommended to spike PhiX into a library prior to loading onto a flow cell?
Follow the manufacturer's instructions for using PhiX. For low-diversity libraries or if one is experiencing sequencing issues, the PhiX concentration may need to be increased. Please refer to the ThruPLEX DNA-Seq Kit User Manual for additional sequencing recommendations.
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