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Amplicon Sequencing Q&A

  • General Questions

  • How do I build a library of amplicons?
    • Amplicon sequencing library building method that mostly uses PCR methods to amplify specific molecular fragments and introduces junction and sequencing primer regions during the amplification process.
  • What is your amplicon library building process?
    • There are currently 2 main process options. One-step library construction means that amplification and ligation are performed simultaneously through one round of PCR reactions. The two-step library construction involves two rounds of PCR reactions with separate amplification as well as ligation.
  • What are the main problems that can be solved by amplicon sequencing?
    • It is currently the most widely used in the field of microbiology, which is used to detect the composition of the microbial community. It mainly includes 16S rDNA sequencing, 18S rDNA sequencing, ITS sequencing and target region amplicon sequencing. The sequences of a highly variable region of 16S/18S/ITS determined by the second-generation high-throughput sequencing platform are used to respond to the differences between species in the taxonomy of bacteria, fungi, and archaea in environmental samples, which is an important guide to study the microbial composition in marine, soil, and intestinal feces environments.
  • Can amplicon sequencing obtain all sequences of the genome?
    • Amplicon sequencing can only detect one or a few specific gene fragments in a sample, but not all genes. The library building process undergoes PCR enrichment and screening, which amplifies the fragments of interest to the researcher tens of thousands and hundreds of thousands of times.
  • What are the factors that affect amplicon library construction?
    • The amplicon library building process is affected by many factors such as amplification method, enzyme, and the number of amplification cycles. The higher the starting concentration of the sample, the higher the number of cycles, and the farther the results deviate from the real situation.
  • Can amplicon sequencing be used for species composition analysis?
    • There are various forms of amplicon sequencing results that can be used to show the composition of bacterial communities in a sample. Venn diagrams, community structure component maps, cluster maps, Heatmap maps, phylogenetic trees, etc. are all good for showing species composition.
  • In what areas can amplicon sequencing be applied?
    • Amplicon sequencing is a type of target region capture sequencing (another target region capture technique is hybridization probe capture). Amplicon sequencing is the sequencing of a specific length of PCR product fragment to analyze the variation in the sequence. Amplicon sequencing is capable of sequencing target regions with high coverage and also detecting low-frequency mutations depending on different needs. The current types of amplicon sequencing include sequencing of functional genes, sequencing of targeted sequence targeted capture fragments, and sequencing of methylated fragments.
  • What is our amplicon sequencing data analysis service?
    • • QC, experimental design, double-ended sequence merging
      • Extraction of barcode, QC and sample splitting, excision of amplification primers
      • Format conversion, redundancy removal, clustering
      • Removal of chimeras, non-bacterial sequences, generation of representative sequences and OTU tables
      • Species annotation, OTU table manipulation
      • Evolutionary tree, Alpha and Beta diversity
      • Species classification statistics, filtering evolutionary trees and others

  • Sample Preparation

  • Do I need to standardize the starting material for my amplicon sequencing?
  • Why do I have high DNA concentrations before I submit and low concentrations after I send it to the company for testing?
    • NanoDrop cannot distinguish between dsDNA and ssDNA (oligonucleotides and dNTP), which may cause you to overestimate the amount of dsDNA in your sample. At our company, we combine Qubit, Nanodrop and agarose electrophoresis to test the quality of DNA samples.
  • How do I purify amplicons?
    • Purified samples can be purified by DNA binding beads/columns, enzyme cleanup and gel purification.
  • What are the sample submission guidelines for amplicon sequencing?
    • • For amplicon sequencing
      Sample Type: Purified Amplicon
      Recommended quantity: ≥ 1 µg
      Minimum quantity: 500 ng
      Minimum concentration: 20 ng/µL
      • For 16S/18S/ITS Sequencing
      Sample Type: Genomic DNA
      Recommended quantity: ≥ 100 ng
      Minimum quantity: 500 ng
      Minimum concentration: 1 g/µL
      Check our Sample Submission Guidelines for details.
For Research Use Only. Not for use in diagnostic procedures.
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