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Exome Sequencing Q&A

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  • General Questions

  • Is it necessary to have a reference genome for whole exome sequencing?
    • Yes, if the reference genome is not available, the sequence of the closely related species should be provided, but the reliability of the capture results cannot be guaranteed. Because the capture probe is designed based on the reference sequence provided, it is not recommended if the target region is known to have a large discrepancy with the reference genome ratio, such as insertional deletion of a large fragment.
  • Why exon sequencing needs to be compared with the whole genome in the analysis, rather than directly with the target region?
    • First, the target region is generally short relative to the whole genome and may be discontinuous, and if the target region is extracted separately, it will affect the sequence comparison effect at the edge of the region. Second, it is impossible to assess the capture quality, such as off-target rate, on target ratio, etc.
  • How many times coverage is generally recommended for whole exome sequencing?
    • Generally, 100× or 150×. Higher coverage ploidy, for measuring heterogeneous genetic variants, can detect a small percentage of mutations. In addition, the coverage of exome sequencing is random, so that a higher average coverage helps to ensure that most of the regions have sufficient coverage ploidy.
  • What is the significance of whole exome sequencing depth? How is the sequencing depth converted?
    • The sequencing depth represents the number of times the sequence is covered by the probe set. The higher the number, the more accurate the identification of sequencing results and the more accurate the subsequent statistical analysis. If doing tumor and low frequency mutation studies, it is recommended that the sequencing depth should be at least 150× or more. If you only look at classical SNPs and non-low-frequency mutations, the sequencing depth should be at least 30× or more. Sequencing depth conversion method: the general target region capture efficiency is 60-70%, the target region size of exon capture is around 60Mb, i.e. sequencing depth = 10G*60%/60Mb = 100×.
  • How large a fragment deletion can be detected by whole exome sequencing?
    • Roughly 50bp of fragment deletions can be detected. Since the coverage of exome sequencing is very uneven, if there are large deletions, it is impossible to tell if it is because the hybridization did not capture them or because they are missing. What can be measured so far is the deletion found in a read. The length of a read is also 150bp, so fragment deletions below 50bp can be measured from exon sequencing.
  • Can whole exome sequencing be used for CNV analysis? What other methods are available for CNV detection?
    • Whole exome sequencing has a hybridization capture process, so there is a problem of hybridization capture efficiency. The hybridization efficiency of each exon is different, and its homologous competition is also different, so the coverage of different exons is very different. Therefore, in general, exon sequencing cannot be used for the detection of CNV. However, in cancer research, CNV can be detected using cancerous and paracancerous tissue controls. there are two other conventional methods to detect CNV, one is whole genome resequencing and the other is with SNP microarrays.
  • Can whole exome sequencing be used for target region methylation detection? Can RNA capture be performed?
    • Target region methylation detection can be performed directly using a methylation capture sequencing product. The principle is similar to target capture sequencing, in which a specially designed probe is used to capture and enrich the target region of interest to the investigator, and then sequenced and methylation detected after bisulfite treatment.
  • Can you capture samples with a multi-species mix?
    • Yes, as long as the target fragment captured has a corresponding reference genome, it can be captured by the probe. Examples include viral sequences integrated in the host genome, parasitic sequences mixed in blood, low abundance microbial sequences in environmental samples, etc. Since the proportion of these sequences in a mixed sample is often very low, the efficiency of using traditional resequencing methods can be very low, requiring very high sequencing volumes to obtain sufficient depth of coverage, while generating a large amount of redundant data. Target capture sequencing has a definite advantage in this regard.
  • Is there an indicator for the effectiveness of exon capture?
    • Due to the limitations of sequencing technology, there are some duplicate sequences, undetermined "N bases", sample quality and other factors that may not be covered.
  • Can exon capture be performed on fragments with high GC content?
    • Yes, but there will be some impact on the capture efficiency of these fragments. Similarly, low-complexity fragments and fragments with ambiguous bases are also difficult to capture. When designing the probes, Euromonitor technicians will increase the number of coverage and probe density according to the coverage, and send the expected capture results to the customer for confirmation. In addition, for capturing whole exons, UTRs, etc., pre-defined probe sets can be used. These probes are designed to optimize the fragments that are more difficult to capture and improve the corresponding capture efficiency.
  • What is exon capture efficiency?
    • The exon sequencing process involves a hybridization process. There are many parts of the human chromosome that are homologous to exons, and these homologous parts are likely to be captured in the hybridization process as well. We call the ratio of the part of sequenced exons to all sequenced sequences the capture efficiency. The capture efficiency does not affect the quality of the data, but only the effective proportion of the data.
  • When to choose full exon capture? When to choose a custom probe capture?
    • There are pre-defined probe products for both human and some common species for whole exon capture, with several versions of the human whole exon probe optimized. If the target region is large and all exons (tens of Mb or more), it is recommended to choose whole exon capture directly, as whole exons are better captured with a more maturely designed probe set and lower cost. For smaller target fragments, or for regions beyond exons that are also of interest, custom probes are recommended.
  • Can I do exon capture sequencing for conventional species, species with new, imperfect or error-ridden genomes?
    • Yes, there is no species restriction on the target capture sequencing platform, only the genome sequence information and the target region information are required. Even if the reference genome of a species is relatively new, inaccurate, or even very different, capture sequencing can be designed. However, accordingly, the capture probes are designed based on the sequences provided by the customer, and therefore the accuracy of the results cannot be fully guaranteed.
  • Can degraded samples be subjected to post-banking? What are the implications for subsequent data and analysis?
    • If the samples are severely degraded, it is not recommended to build a library at risk and the success rate of building a library is low; if the samples are severely degraded, the impact of building a human exome library is mainly low capture efficiency, low effective data volume and low properly mapped.

  • Sample Preparation

  • What is the impact of sample viscosity, contamination by micellar impurities and RNA contamination on library construction? What is the approximate success rate of library construction?
    • However, if the contamination of protein and other impurities is serious, it will affect the quantification and the enzyme efficiency in library building, which will reduce the success rate of library building; RNA contamination mainly affects the quantification of samples and the sorting of DNA in library building; therefore, if RNA contamination exists, RNA digestion is recommended if the total amount and quality of samples are suitable.
  • Can a single piece of tissue be co-extracted for DNA and RNA? What is the extraction method?
    • There are two methods of co-extraction, one is to cut the tissue into two parts and extract DNA and RNA each; the other is to use a DNA/RNA co-extraction kit to extract, but usually the DNA and RNA are easily degraded after extraction.
  • What are the reasons for the low DNA extraction rate of FFPE samples?
    • FFPE samples are usually stored for many years and have been severely degraded by formaldehyde; and FFPE samples are usually precious, so teachers send less samples.
  • How to separate plasma samples for cf/ctDNA extraction?
    • Collect 5 mL of peripheral blood (usually collected in the morning or early morning), quickly transfer to an EDTA anticoagulation tube, carefully invert and mix (to prevent hemolysis); separate plasma within 1 hour (at room temperature) or 2 hours (at 4°C): centrifuge at 1,000 rpm for 10 minutes at 4°C, carefully aspirate plasma into a clean 1.5 mL EP tube (be careful not to aspirate the plasma into Then centrifuge at 12,000 rpm for 10 minutes at 4°C to remove residual cells or debris, carefully aspirate the desired volume of supernatant into a new EP tube, mark with an oil-based marker pen, store in an ultra-low temperature refrigerator at -80°C, avoid repeated freeze-thawing, and transport the sample on dry ice.
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