◆ General questions about our DNA-seq kits
(1) Amplification 반응을 위해 정확성이 높은 PCR효소 (High-fidelity PCR enzyme)가 필요한가요?
◆ Questions about specific DNA-seq kits
Is a high-fidelity enzyme used in the amplification reaction?
(2) DNA-Seq Kit에 사용 가능한 Thermal Cycler장비는 무엇인가요?
Yes, a high-fidelity, high-processivity, low-bias DNA polymerase is used in our DNA-seq kits.
Can the NGS DNA-seq kits be run on any thermal cycler?
(3) DNA-Seq Kit 사용 시, 추가로 필요한 제품은 무엇인가요?
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 NGS DNA-seq kits?
(4) DNA-Seq Kit 내에 포함된 모든 시약의 혼합 시, vortexing을 해도 되나요?
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 NGS DNA-seq kits be mixed by vortexing?
(5) DNA-Seq Kit을 methylation 연구에 적용할 수 있나요?
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 NGS DNA-seq kits be used for methylation studies?
(6) DNA-Seq Kit으로 제작한 Library를 microarray나 PCR 분석용으로 사용할 수 있나요?
Can libraries created using NGS DNA-seq kits be used for microarray or PCR analyses?
(7) PCR 증폭 (PCR amplification) 없이 Library 제작이 가능한가요?
Yes, the libraries created using our DNA-seq kits can be used for microarray and PCR analyses.
Can a library be prepared without any PCR amplification?
(8) Additional amplification 단계 전의 Library 증폭 산물을 보관 가능한가요?
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?
(9) NGS DNA-seq Kit은 single index를 사용하나요? Dual index를 사용하나요?
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 NGS DNA-seq kits use single or dual indexes?
(10) NGS DNA-seq Kit의 보관 조건은 무엇인가요?
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.
What are the storage conditions for the NGS DNA-seq kits?
(11) NGS DNA-seq Kit 보존 기간은 어떻게 되나요?
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 the NGS 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.
1. PicoPLEX Gold Single-Cell Kit에 대한 FAQ
Questions about the PicoPLEX Gold Single-Cell Kit
(1) PicoPLEX Gold Single Cell DNA-Seq Kit은 어떤 실험에 적합한가요?
What applications are recommended for the PicoPLEX Gold Single Cell DNA-Seq Kit?
(2) Single cell의 whole genome de novo sequencing 목적으로 사용해도 되나요?
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?
(3) Library 제작 소요시간은 얼마나 걸리나요?
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?
(4) 적용 가능한 세포의 종류는 무엇인가요?
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?
(5) 항체로 염색한 세포를 샘플로 사용해도 되나요?
Single blastomeres, trophectoderm cells, and cultured cells have been used successfully.
Can cells stained with surface antibodies be used as input?
(6) NGS index를 별도로 구매해야 하나요?
Yes, cells stained with surface antibodies can be used if the cells are not fixed.
Do we have to purchase barcoded oligonucleotides separately?
(7) 실험실에서 직접 제작한 index를 사용해도 되나요?
Can we use barcodes designed in our laboratory?
(8) Kit에 적용가능한 Index는 무엇인가요?
What are the indexing options for the PicoPLEX Gold Single Cell DNA-Seq Kit?
(9) Sequencing read 분석 시, base trimming이 필요한가요?
How many bases should I trim for analysis of the sequencing reads?
(10) Single-end, Paired-end sequencing에 모두 사용할 수 있나요?
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 I prepare samples for both single- and paired-end NGS sequencing?
(11) Ion Torrent 또는 PacBio 플랫폼에 적용 가능한가요?
Libraries prepared using the PicoPLEX Gold Single Cell DNA-Seq Kit are compatible with both single- and paired-end sequencing on Illumina platforms.
Can the PicoPLEX Gold Single Cell DNA-Seq Kit be used with Ion Torrent or Pac Bio platforms?
(12) PicoPLEX Gold Single Cell DNA-Seq Kit로 제작한 Library의 크기는 어떻게 되나요?
No, this kit is not compatible with Ion Torrent or Pac Bio.
What is the size range of fragments expected with libraries prepared using the PicoPLEX Gold Single Cell DNA-Seq Kit?
(13) 사용 가능한 Thermal Cycler장비는 무엇인가요?
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?
(14) Real Time PCR 장비 없이 사용 가능한가요?
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?
(15) Illumina sequencer적용 전, Library 정량은 어떻게 하나요?
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.
How do I quantify the pooled, purified library for loading onto an Illumina sequencer?
(16) Illumina MiSeq flow cell에 로딩 시, 필요한 library 농도는 얼마나 되나요?
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?
(17) Library 정제 시, AMPure XP beads의 적정 비율은 어떻게 되나요?
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?
(18) PicoPLEX Gold Single Cell DNA-Seq Kit의 GC coverage는 어떻게 되나요?
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?
>90% of the reads from libraries generated with the PicoPLEX Gold Single Cell DNA-Seq Kit fall between 25% and 70% GC content.
2. Questions about the PicoPLEX Single-Cell WGA Kit
(1) PicoPLEX WGA의 강점은 무엇입니까?
What are the strengths of PicoPLEX versus other WGA technologies?
(2) 적정 DNA input양 및 샘플 가이드라인은 어떻게 되나요?
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?
(3) 적용 가능한 세포의 종류는 무엇입니까?
- 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)
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?
(4) Cell collection시, washing을 해야 하나요?
Single blastomeres, polar bodies, trophoblasts, amniocytes, and cultured cells have been amplified successfully.
Should cells be washed before collection?
(5) Single cell washing을 위한 nonstick wash buffer가 있나요?
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?
(6) PicoPLEX NGS kit에 적합한 cell collection 방법은 무엇인가요?
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?
(7) Flow sorting 시, 주의사항이 있나요?
Flow sorting, dilution, and micromanipulation are compatible with PicoPLEX NGS kits.
Are there special requirements for flow sorting?
(8) PicoPLEX Single Cell WGA Kit의 input sample의 양은?
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?
(9) Sigma-Aldrich가 판매 중인 GnomePlex WGA Kit과 원리가 같나요?
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?
(10) PicoPLEX Kit로 Whole Genome Amplification에 소요되는 시간은?
How rapid is the PicoPLEX Single Cell WGA Kit?
(11) PicoPLEX Single Cell WGA Kit의 프로토콜이 좋은 점이 무엇인가요?
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?
(12) 재현성은 어느 정도인가요?
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?
(13) PicoPLEX Single Cell WGA Kit 산물의 genomic locus representation과 Loss of Heterozygosity (LOH)가 어떻게 되나요?
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?
(14) Buffer control 실험의 결과와 single cell 실험의 결과를 구분 가능한가요?
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
Illumina Infinium SNP array
Can buffer control reactions be distinguished from single-cell reactions?
(15) PicoPLEX WGA Kit의 산물은 single-strand DNA인가요, double-strand DNA인가요?
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?
(16) PicoPLEX Single Cell WGA Kit에 적합한 실험(application)은 무엇인가요?
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?
(17) SNP Genotyping에 적합한 이유는 무엇인가요?
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?
(18) 본 제품으로 gel-based STR genotyping을 할 수 있나요?
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?
(19) Probe-based qPCR 분석이나 sequencing assay에 적용 가능한가요?
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?
(20) 본 제품의 산물을 microarray 적용하고자 할 때, label은 어떻게 하나요?
Yes, as long as the amplimers are less than 250 bp.
How can I label PicoPLEX Single Cell WGA Kit products for microarrays?
(21) qPCR assay 또는 array에 적용할 때, PicoPLEX Single Cell WGA Kit 산물이 어느 정도 필요한가요?
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?
(22) PicoPLEX Single Cell WGA Kit로 제작된 산물을 NGS에 바로 적용 가능한가요?
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?
(23) PicoPLEX Single Cell WGA Kit로 제작된 산물을 NGS-ready 상태로 만들 수 있나요?
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?
(24) 다카라바이오는 Single cell DNA-Seq을 위한 제품을 제공하나요?
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?
3. Questions about the ThruPLEX DNA-Seq Kit
(1) ThruPLEX DNA-Seq Kit의 강점은 무엇인가요?
What is the difference between the ThruPLEX DNA-Seq Kit and other commercially available library preparation kits?
(2) ThruPLEX DNA-Seq Kit의 GC-rich 영역에도 적용 가능한가요?
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?
(3) Denatured DNA 또는 single stranded DNA도 적용 가능한가요?
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?
(4) DNA 단편화 (DNA fragmentation)를 꼭 해야 하나요?
No, DNA must be double-stranded for use with the ThruPLEX DNA-Seq Kit.
Is DNA fragmentation necessary?
(5) 권장하는 DNA 단편화 방법은 무엇인가요?
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?
(6) Covaris shearing을 이용할 때 필요한 DNA의 양과 버퍼의 종류는 무엇인가요?
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?
(7) DNA 단편화 시, 권장하는 Covaris shearing 사양은 무엇입니까?
What Covaris shearing specifications should be used to fragment the DNA prior to ThruPLEX DNA-Seq Kit usage?
(8) DNA 단편화를 위한 Random fragmentation을 이용하고자 한다면, 권장 사항은 무엇입니까?
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?
(9) 권장하는 DNA 샘플양 (DNA input range)이 어떻게 되나요?
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?
(10) Indexing 시약이 꼭 필요한가요? 만약 그렇다면, Multiplexing 시에 어떤 index를 써야 하나요?
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?
(11) Library amplification 반응에 필요한 적정 PCR Cycle 수를 어떻게 정하나요?
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?
(12) Real Time PCR 장비 없이 사용 가능한가요? 일반 PCR 장비를 사용한다면 PCR 반응 조건은 어떻게 되나요?
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?
(13) Library 산물의 수율 또는 농도가 충분하지 않을 경우, 추가로 amplification step을 진행해도 되나요?
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?
(14) AMPure XP bead로 정제를 완료한 Library 산물을 이용해 amplification step 추가로 진행해도 되나요?
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?
(15) NGS Library의 제작 수율은 어떻게 되나요?
What is the expected yield for the amplified library from the ThruPLEX DNA-Seq Kit?
(16) Library 정량은 언제 해야 하나요?
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?
(17) Library를 AMPure XP Cleanup 단계 전, 후 모두 정량을 해야만 하나요?
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?
(18) AMPure XP purification 진행 시, 준수해야하는 주의사항이 있나요?
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?
(19) 어떤 Illumina NGS 장비를 사용 가능한가요?
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?
(20) Flow cell에 로딩 시, 어느 정도의 library 농도가 필요한가요?
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?
(21) Flow cell 로딩 시, 꼭 준수해야만 하는 특별한 단계가 있나요?
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?
(22) Flow cell 로딩 시, PhiX spike-in control을 꼭 써야만 하나요?
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.