In the previous NGS series of lectures, we invited experts to introduce you to the comprehensive NGS technology application from sample preparation to data interpretation report. The lecture received a lot of feedback and consultation, here we summarize the 7 most concerned issues, and share with you.
1 Which NGS platforms can QIAGEN's targeted gene amplification kits be used for?
GeneRead DNAseq Targeted Panels V2 is available for Illumina® and Ion Torrent® sequencers. After targeted enrichment, you can perform sequencing library preparation using the QIAseq 1-Step Amplicon Library Kit (for the Illumina sequencer) or the GeneRead DNA Library Core L Kit (for the Ion Torrent sequencer) depending on the sequencing platform used.
2 What is the minimum sequencing depth recommendation for DNA biopsy cfDNA sequencing using DNA targeting technology?
For the identification of sites for somatic mutations, we recommend a sequencing depth of 1500x.
3 What is the difference between the PCR enrichment method and the capture enrichment method? Which method should I use?
Chip or hybrid capture methods are suitable for longer sequencing targets. The PCR enrichment method is suitable for target enrichment with a target length of about 1 Mb. So you need to choose an enrichment method based on your research needs. The PCR method is more specific, more uniform, and more reproducible.
PCR enrichment | Hybrid enrichment | |
Specificity | 93% | 80% |
Uniformity | 80% | 61% |
Repeatability | 100% | 95% |
4 What types of variation can QIAGEN's enrichment method detect?
You can detect point mutations, insertion/deletion mutations, and copy number changes.
5 Can I customize the panel only for the sites I need to study?
Yes, you can use the GeneRead DNAseq Custom Builder to customize the multiplex PCR targeting enrichment panel you need for your experiment.
6 How much DNA can I get from a liquid biopsy sample?
About 1–50 ng of free circulating DNA can be obtained from 1 ml of plasma. Sample types (such as tumor samples or non-tumor samples), sample collection procedures, storage and processing methods have an impact on yield.
7 Does the amplicon usually cover all exon regions of the gene, or does it only cover the hotspots that need to be studied?
Both situations are fine. Most panels cover all exon regions of the target gene, while the human tumor actionable mutation panel covers only the relevant hotspot regions that need to be studied.
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