FAQs

Questions for DNA Library Construction

• What are the methods can I use for library quantification?
  • There are two methods for library quantification: Qubit and qPCR, used for determining library mass concentration and library molarity, respectively. qPCR can truly reflects the number of DNA fragments used for machine sequencing owing to the theory of clustered primers performing amplification quantification. Therefore, the library quantitative results measured by qPCR are more reliable. These two methods can be used at the same time to quantify libraries and correct each other.
• What are the possible reasons for low library yields?
  • 1. Insufficient amount of starting DNA. Make sure to use an accurate DNA quantification method to ensure sufficient DNA input. Try increasing the amount of starting DNA.
    2. Suboptimal reaction conditions due to low DNA quality. Make sure to use the highest quality sample DNA available, and try to increase the cycles according to the protocol.
    3. Wrong reaction volumes or conditions used for end repair, ligation or PCR amplification. Make sure to use the exact conditions in the protocol.
    4. Overdrying of the clean beads during cleanup steps. Overdrying of the clean beads can make it difficult to elute the DNA off the beads.
    5. Lower than expected library yield with extensive adaptor dimer. Excessive adaptor dimers can compete with the amplification of the real libraries during library amplification PCR step and lead to low yield of specific libraries. Make sure the right dilution of the adaptor is used. Make sure the correct clean beads purification protocol is used.
• Unexpected broad peaks spread across 500 – 10,000 bp range.
  • 1. There are nonspecific products due to overamplification. Double check that the correct PCR cycle numbers were used. PCR cycles are determined based on the DNA input amount and DNA quality. Try reducing the amplification cycles if issue persists.
    2. If the fragment population shifts higher than expected, this can also be due to the carry-over of the clean beads. Make sure not to aspirate beads while taking supernatant during the cleanup steps.
• Presence of shorter peaks between 60 and 120 bp and the implications for sequencing.
  • These peaks represent library adapters and adapter dimers that occur when there is no, or insufficient, adapter depletion after library preparation. As adapter dimers can form clusters on the Illumina flow cell and will be sequenced, this will reduce the capacity of the flow cell for the library fragments. A low ratio of adapter dimers versus library will not be a problem. Please make sure the correct dilution of the adaptors and the correct volume of the clean beads is used for the cleanup steps.

Questions for RNA Library Construction

• What are the methods can I use for library quantification?
  • There are two methods for library quantification: Qubit and qPCR, used for determining library mass concentration and library molarity, respectively. qPCR can truly reflects the number of DNA fragments used for machine sequencing owing to the theory of clustered primers performing amplification quantification. Therefore, the library quantitative results measured by qPCR are more reliable. These two methods can be used at the same time to quantify libraries and correct each other.
• Is FFPE samples suitable for library construction by CD mRNA-seq Library Prep Kit/CD Stranded mRNA-seq Library Prep Kit?
  • It is recommended to use CD Total RNA-seq Library Prep Kit for Illumina (human/mouse/rat) / CD rRNA depletion kit(Plant)/ CD rRNA Depletion Kit (Bacteria) for FFPE sample, as the mRNA in FFPE sample typically have been degraded and with poor integrity.
• Is the CD mRNA-seq Library Prep Kit/CD Stranded mRNA-seq Library Prep Kit suitable for non-eukaryotic mRNA library construction?
  • Because the kit isolate mRNA via magnetic beads with Oligo dT directly capturing mRNA, it is not suitable for non-eukaryotes without poly-A.
• Is the CD mRNA-seq Library Prep Kit/CD Stranded mRNA-seq Library Prep Kit/ CD Total RNA-seq Library Prep Kit (human/mouse/rat) suitable for small RNA library construction?
  • Not applicable. Considering the length of small RNA is only about 22nt and our clean beads purification and size selection at least 100 bp long, this kit cannot efficiently enrich small RNA fragments.
• What methods can I use to solve the problem of low concentration of library by CD mRNA-seq Library Prep Kit/CD Stranded mRNA-seq Library Prep Kit?
  • It is recommended to use high-quality RNA samples as templates for library construction to make library concentration meet the requirements of the machine sequencing. If you cannot provide qualified RNA samples, try to use the following methods to make up:
    1. Initial amount: Increase the initial amount to 10ug;
    2. Do several duplicate samples, merge them at the two-strand synthesis purification step, or before PCR step.
    3. Take option without sorting: Though RNA fragmented at 94°C for 8 min is short, its distribution is concentrated and the homogeneity is also well. However, some individualized samples have non-uniform fragments and this situation will be amplified by PCR, but this situation rarely occurs in the option of without sorting.
    4. If the sample have been degraded and with poor integrity, use CD Total RNA-seq Library Prep Kit for Illumina (human/mouse/rat) / CD rRNA depletion kit(Plant)/ CD rRNA Depletion Kit (Bacteria) instead.
• Instructions for adapter selection.
  • At present, the adapters applicable to CD mRNA-seq Library Prep Kit/CD Stranded mRNA-seq Library Prep Kit/CD Total RNA-seq Library Prep Kit for Illumina (human/mouse/rat) is divided into three sets:
    Set 1: CD RNA Adapters S1-S2 for Illumina (Adapter 1-27, RSI001) totally contains 24 different adapters, divided into two separate packages, each containing 12 different adapters according to the serial number.
    Set 2: CD RNA Adapters S3-S6 for Illumina (Adapter 1-96, RSI002) totally contains 96 different adapters, divided into four separate packages, each containing 24 different adapters according to the serial number.
    The above two sets of adapters cannot be mixed in the same batch of sequencing samples, refer to the following suggestions:
    1. When the number of samples in the same batch of sequencing is < 24, it is recommended to select set 1; When the number of samples is < 12, the individual package of the set can be selected.
    2. When the number of samples in the same batch of sequencing is between 24 and 96, it is recommended to select set 2, but the individual package of this set can also be selected according to the number of specific samples.
    Set 3: CD RNA Multiplex Oligos S1 for Illumina (RDI001) totally contains a Universal Adapter, 8 kinds of 8bp indexed i5 PCR Primers and 12 kinds of 8bp i7 PCR Primers, which can provids libraries with 96 kinds of different dual-index combinations.
• What are the affects for over-amplified high-yield libraries tested on the Agilent 2100 Bioanalyzer, Qubit and qPCR?
  • High-yield libraries are often over-amplified in different degrees. Because at the later period of library amplification, primers are usually exhausted. Therefore, a large number of library fragments cannot be combined with primers, and the fragments are incorrectly annealed through incomplete matching. Thus, a hybrid strand mixed with partial double strand and partial single strand is formed in larger size. According to the corresponding principles of different detection methods, excessive amplified products show slight tailing after the upper marker in the analysis graph of Agilent 2100 Bioanalyzer; the single-stranded portion can not be detected by the Qubit, but can be effectively measured by qPCR, thus the concentration measured by Qubit is lower than that measured by qPCR at about 10% - 50%. The above phenomenon is normal and would not affect the library sequencing and data analy.
• Why there are double peaks in the graph when the library was tested on the Agilent 2100 Bioanalyzer?
  • 1. There are residual impurities and degrades of RNA during library construction; the amount of effective template is low when PCR, causing non-specific amplification. It is recommended to heat RNA sample at 65°C for 15 min for degradation test. If RNA is unqualified, the RNA re-extraction must be taken.
    2. The species itself is special. The RNA fragments are not continuous and uniform after fragmentation, and two ranges of fragments might be selected.
    3. The mRNA abundance of specific species and the effective amount of template for PCR are insufficient. It is recommended to increase the amount of input or to mix repeated samples at the purification step after two-strand synthesis.
• Why the sorting insertions is larger than the actual insert when operating as the handbook?
  • There are various reasons that cause the amount of magnetic beads added less than the specified value, resulting in the larger sorting insertions: the magnetic beads are not equilibrated to room temperature or not mixed thoroughly; the pipette is inaccurate, and the tip of the pipette is severely attached.
• If the purified product is used for library construction, but use Nuclease-free H2O for elution by mistake. What can I do?
  • Using CD RNA Clean Beads (CB002) to purify again, and finally elute with Frag/Prime Buffer.
• How long can the purified product be stored by CD Total RNA-seq Library Prep Kit for Illumina (human/mouse/rat) / CD rRNA depletion kit(Plant)/ CD rRNA Depletion Kit (Bacteria)?
  • The purified product is easily degraded due to its low concentration. It is recommended to proceed to the next procedures immediately after eluting Ribosomal-depleted RNA. Otherwise, store Ribosomal-depleted RNA at -70°C.

Questions for Small RNA Library Construction

• Can centrifugal column be used to extract RNA except TRIZOL?
  • This Kit has no special requirements for RNA extraction. If use centrifugal column to extract RNA, please ensure that the kit used will not cause the loss of small RNA.
• Can RT primer be added at the cDNA synthesis Step?
  • No. RT primers added after ligation of 3'-Adaptor can be complementary reversely with extra 3'-Adaptor to form a double chain structure, which effectively prevents the ligation between 5'-Adaptor and 3'-Adaptor, and reduces the adaptor dimers; Besides, RT primer complements reversely with RNA substrate with 3'-Adaptor can be the primer of reverse transcription, so RT primer must be added after 3'-Adaptor ligation instead of cDNA synthesis Step.
• Can other total RNA be as initial template for library construction except total RNA of plants and animals?
  • Except total RNA of plants and animals and purified small RNA, the Kit is suitable for other total RNA, such as total RNA of exosomes.

Questions for CD DNA Methylation Bisulfite Kit

• Should the input DNA be dissolved in TE, water, or some other buffer prior to its conversion?
  • Water, TE or modified TE buffers can be used to dissolve the DNA and do not interfere with the conversion process.
• What are the possible reasons for low DNA-recovery yield?
  • 1. Poor quality of initial DNA. Ensure that the A260 / 280 ratio of DNA is between 1.7 and 1.9; The DNA bands in gel electrophoresis are clear without degradation.
    2. Insufficient ethanol in E-Wash Buffer. Add the proper volume of absolute ethanol, DO NOT open the lid for a long time.
    3. Insufficient organic solvent in E-Desulphonation Buffer. DO NOT open the lid for a long time.
• What are the possible reasons for low conversion efficiency?
  • 1. CT Conversion Mix is invalid. Please prepare the CT Conversion Mix correctly according to the handbook and use it during its shelf life.
    2. Set the wrong temperature or reaction time. Set the correct temperature and reaction time according to the handbook.
    3. Add excess input DNA, or DNA sample with high GC-content. DNA samples with high GC-content or excess DNA input. Control the Input DNA within optimal range or extend the reaction time of 64°C to 50 min - 60 min.

Questions for CD RRBS Library Kit

• Does the CD RRBS Library Kit offer all the necessary reagents to generate the libraries for Reduced Representation Bisulfite Sequencing?
  • Yes. With this all-in-one kit, you can generate RRBS libraries with the genomic DNA of your interest. However, the reagents for library quantification and size distribution characterization are not provided.
• Is there any restriction on the sample species when using this kit?
  • In principle, the CD RRBS Library Kit can be applied to genomic DNA from any species, as long as a reference genome exists for subsequent bioinformatic analysis and the genomic DNA is between 10 and 500 ng with high quality (a good rule of thumb for "high quality" is that the DNA is of little degradation and the majority of the content being higher than 10 kb).
• Can I use less than 10 ng of genomic DNA with this kit?
  • It is possible to generate libraries with less than 10 ng of genomic DNA; however, the quality of such libraries is not guaranteed.
• Can I use DNA extracted from FFPE samples to generate RRBS libraries with this kit?
  • It is possible to generate libraries with DNA extracted from FFPE samples. However, due to the degradation of such DNA, the quality of the libraries will be affected. Increasing the input amount may help but the quality of the libraries is not guaranteed.
• What method of quantification is recommended for the input DNA?
  • We quantify input DNA using Nanodrop or Qubit, but any method should do. The input genomic DNA should be RNA-free.
• How many libraries can I multiplex and sequence in a single lane using this kit?
  • We provide 12 single-indexed primer sets with the kits and therefore you can multiplex up to 12 libraries.
• What is the expected yield of each library generated using this kit?
  • The yield per library depends on the quality of the input genomic DNA and other factors. In general, the yield of each library is around 300 ng or more (if quantified with Nanodrop) when following the recommended PCR cycles in the handbook.
• What read length and sequence depth should I seek for when sequencing RRBS libraries?
  • The optimal read length and sequence depth depend on the goal of the project and the subsequent bioinformatic analysis. In general, when handling mammalian samples, 50 bp single-end or paired-end sequencing should do as the RRBS libraries usually have comparatively short inserts. Longer read length is also possible if one wants to cover as many CpG sites as possible. Additionally, a 5-10x mean coverage per CpG site is recommended.

Questions for CD scmRNA Amplication Kit

• Is this kit applicable to plant cell samples?
  • No. The lysis buffer in this kit cannot lyse cell wall efficiently. For eukaryotic cells with cell walls, please lyse the cell wall before using this kit, or use purified RNA as template.
• Is this kit applicable to fixed cells?
  • No. After treatment with formaldehyde or acetone, the quality of RNA in tissue or cells declined significantly, cause the failure of amplification. So this kit is not applicable to fixed cells.
• Are cell samples can be operated with this kit directly without rinsed with PBS solution?
  • For cultured cells, please confirm if the culture medium has inhibitory effect on the reaction. It is recommended to add the medium to the RNA control reaction to check whether the medium inhibits the cDNA synthesis. If it is hard to identify, please resuspend the cells in PBS before starting the reaction.
• Could the cell samples be stored for a certain time, when we can't conduct amplification immediately after the samples are prepared?
  • If your sample needs to be stored for a certain time, please refer to the Section 09-1/ Step 2 and Step 3 for operation. The prepared cell samples should be stored at -70°C or lower temperature, and please start the reaction immediately after taking out from refrigerator. The isolated living cells could be stored in freezing medium, and recovered before use. Please check if the cells stay alive before reaction, because dead cells showing obvious RNA degradation may cause the failure of the experiment.
• How to confirm the cycle number when amplify full-length cDNA through PCR?
  • Select the optimal cycle number to ensure that the amplification is still in the exponential phase. When the expansion cycle number increases, while cDNA yield no longer increase accordingly, the amplification has reached a plateau. Over-amplified cDNA will lead to a decline in quality of cDNA library. However, low cycle number will cause a decline in cDNA yield. Therefore, please set the cycle number as low as possible on the premise of yielding enough products. It is recommended to set several parallel reactions with different cycle numbers to determine the optimal cycle number. For example, set up three reactions, one reaction carried out in accordance with the recommended cycle number, the other two reactions with 2-3 less cycles and 2-3 more cycles respectively.
• How is the single-cell transcriptome amplification yield? And how to prepare the library for sequencing?
  • The gene expression in different cells varies instantaneously. Cell type, activity, and cycle will significantly influence the final cDNA yield. Under normal circumstances, CD scmRNA Amplication Kit can output 2-20 ng of cDNA library. It is recommended to prepare library using transposase method: take 1 ng of cDNA as starting material, and use CD NEXT DNA Library Prep Kit for Illumina (DP002) or Nextera® XT DNA Library Preparation Kit (Illumina, #FC-131-1024) to prepare library for sequencing.

Questions for CD scWGS MDA Amplication Kit

• Could the cell samples be stored for a certain time, when we can’t conduct amplification immediately after the samples are prepared?
  • If your sample needs to be stored for a certain time, the isolated living cells could be stored in 4ul of PBS at -70°C or lower temperature, and please start the reaction immediately after taking out from refrigerator.
• 10-40 μg of DNA is amplified but loss some loci or alleles.
  • A. For reaction of using genomic DNA as initial template: the genomic DNA may be degraded, please use complete DNA or more amount of DNA as template.
    B. For reaction of using cell as initial template: cell apoptosis or DNA degraded during the fixed step. Or there is cell wall (i.e. plant cells) which is not suitable for direct initial material.
• 10-40 μg of DNA was amplified in the negative control but the downstream detected results were negative (i.e. qPCR).
  • The high molecular weight DNA products were amplified by the random annealing of primers in the negative control reactions, but the products do not affect downstream analysis of the target product.
• 10-40 μg of DNA was amplified in negative control and the downstream detected results were positive (i.e. qPCR).
  • The reaction is contaminated with exogenous DNA. Since the reaction is very sensitive to micro DNA, please replace all the reagents and supplies that may be contaminated with exogenous DNA.

Questions for CD Library Quantification Kit for Illumina

• What are the possible reasons for amplification efficiency beyond the range of 90% - 110%?
  • 1. The qPCR reaction system is contaminated if Ct (NTC) - Ct (DNA Standard 6) < 3 or Ct (DNA Standard 6) - Ct (DNA Standard 5) < 3.1, while the calculated amplification efficiency is above 100%. Determine the source of contamination (Standard DNA or library DNA) by examining the melt curves of NTC.
    2. Inappropriate Baseline setting may delay Ct value of DNA Standard 1, further affecting the calculation of PCR efficiency. Manually adjust the baseline to 1-3 cycles.
    3. Poor accuracy in liquid handling.
• The R² value of the standard curve is < 0.99.
  • 1. Poor accuracy in liquid handling.
    2. All reagents should be thawed and mix thoroughly before use.
    3. Ensure that the appropriate ROX reference dye was used.
• The amplification curve of standards are not evenly distributed.
  • 1. If Ct (DNA Standard 6) - Ct (DNA Standard 5) < 3.1, the reaction system is contaminated. Determine the source of contamination (Standard DNA or library DNA) by examining the melt curves of NTC.
    2. If Ct (DNA Standard 2) - Ct (DNA Standard 1) < 3.1, the baseline setting inappropriate. Manually adjust the baseline to 1-3 cycles.
    3. If ΔCt among DNA Standards is > 3.6, the amplification efficiency is poor. Ensure that all reagents are thoroughly thawed and mixed before use. Confirm that all reaction components were added at the correct concentration, and that the correct cycling protocol was used.
    4. Long time exposure in light of SYBR qPCR Master Mix will reduce total fluorescence and may result in ΔCt values >3.6.
• Poor reproducibility between replicates.
  • 1. Poor accuracy in liquid handling.
    2. All reagents should be thawed and mix thoroughly before use.
    3. Ensure that the appropriate ROX reference dye was used.
• ΔCt of library dilutions is not within expected range.
  • 1. Poor accuracy in liquid handling.
    2. All reagents should be thawed and mix thoroughly before use.
    3. The library is difficult to amplify, i.e. the library is extremely GC- / AT-rich or has a long average fragment length (>1 kb).
    4. The library has degraded. Prepare fresh dilutions of library and keep on ice during reaction setup.
• Concentrations calculated from different library dilutions differ by more than 10%.
  • 1. Poor accuracy in liquid handling.
    2. All reagents should be thawed and mix thoroughly before use.
    3. The library is difficult to amplify, i.e. the library is extremely GC-/AT-rich or has a long average fragment length (>1 kb).
    4. The library has degraded. Prepare fresh dilutions of library and keep on ice during reaction setup.
• Library dilutions fall outside of effective range of standard curve.
  • 1. If Ct (diluted library) < Ct (DNA Standard 1), the library dilution is not sufficient, usually happening in over-amplified libraries. Increase library dilution factor and repeat quantification reaction.
    2. If Ct (diluted library) > Ct (DNA Standard 6), the library is over-diluted or the library preparation fails. The Ct value of a conventional library with a dilution factor around 1:10,000 should not exceed the Ct value of DNA Standard 6. Decrease the library dilution factor and repeat quantification reaction.
• The Ct of DNA Standard 1 appears abnormal.
  • 1. Inappropriate Baseline setting may delay Ct value of DNA Standard 1, further affecting the calculation of PCR efficiency. Manually adjust the baseline to 1-3 cycles.
    2. Ensure that the appropriate ROX reference dye was used.
• Samples of DNA Standards is amplified, but sample of libraries are not. Or significant difference in Ct between DNA Standards and Library Samples.
  • 1. The library molecules does not contain the appropriate adapter sequences for the quantification primers to bind to.
    2. The library is over-diluted. Decrease the library dilution factor and repeat quantification reaction.
    3. The library has degraded. Prepare fresh dilutions of library and keep on ice during reaction setup.