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Degradome Sequencing Q&A

  • General Questions

  • Are there any species for which degradome sequencing is applicable?
    • Unlike animals, plant miRNAs usually undergo complete or near-complete pairing with target genes to cause target gene shearing and thus regulate gene expression, so there will be fewer false positives and more accurate identification results. Compared to animals, plants are more suitable for studying miRNA target genes using degradome sequencing. In contrast, model organisms or species with detailed transcriptome information are more suitable for miRNA target genes using degradome sequencing than species with incomplete transcriptome information.
  • What is the minimum starting volume for degradome sequencing samples?
    • The recommended starting volume for degradome sequencing is > 20 μg total RNA. For plant tissues, 1 g or more of fresh tissue is recommended (there are some differences between different tissue sites).
  • How much data volume should be sequenced for degradome sequencing?
    • Generally, 10M reads are sufficient for conventional species. For species with large and complex genomes, the amount of sequencing data can be doubled.
  • How many biological replicates are required for degradome sequencing?
    • Biological replicates are recommended to be consistent with the number of miRNAs (≥3), and special cases can be considered by mixing the samples within the group and adding the data volume.
  • What can degradome sequencing do besides detecting the target genes of miRNAs?
    • First of all, we need to understand that one of the biggest roles of degradome sequencing is to find the target genes of miRNAs, but of course, as researchers analyze degradome data in depth, they find that it can also be used to study miRNA self-regulation, ta-siRNA, phasiRNA, processing of precursor sequences, and for detecting new miRNAs.
  • What are the sequencing platforms and read lengths used for degradome sequencing?
    • Both Illumina and MGI are currently available as mainstream second-generation sequencing platforms. Due to the short target fragments, SE50 sequencing strategy is used.
  • What is the difference between degradome sequencing and RACE validation?
    • Although raw signal prediction can predict the target genes of miRNAs with high throughput, there are a lot of false positives and the results are not reliable. RACE validation is time-consuming and labor-intensive, and does not have the same high throughput as raw signal prediction. Therefore, the advantage of degradome sequencing is that not only the target genes of miRNAs can be detected in high throughput, but also the results are obtained by biological experiments, which greatly improves the credibility of the experimental results.
  • What is the exact meaning of Alignment Score in the result?
    • Alignment Score is a score for the prediction of target genes by applying targetfinder to the cDNA library and small RNA library of the sequenced species. miRNA and mRNA are compared and scored as complementary pairings (1 score for a mismatch or deletion, 0.5 score for a G:U pairing, double score for mismatch and G:U pairing in the core region, starting from the 2nd nt to the 13th nt of microRNA). The alignment score is defined as the degree of match between target gene and miRNA, the lower the score the more complete the match is and the more reliable it is.
  • What is the specific meaning of Category in the result?
    • The significance of Category typing is to visualize the number of degraded fragments produced by miRNA cutting mRNA, and the confidence level is Category 0>1>2>3>4.
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Degradome Sequencing
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