T/LTIA 26-2024 Species identification technology specification based on SeqFD technology

Scope

Scope: This document specifies the principles, reagents or materials, instruments and equipment, experimental procedures, and result analysis for species identification based on SeqFD sequencing technology. This document is applicable to plant and animal samples related to agriculture, such as rice, cassava, sugarcane, potatoes, pigs, and cattle, etc. Main technical content: Design primers containing degenerate bases, construct sequencing libraries for sample genomes, perform library amplification and high-throughput sequencing using multiplex polymerase chain reaction (PCR), analyze sequencing data, obtain alignment results and identification conclusions. By using primers containing degenerate bases, construct sequencing libraries for sample genomes, and perform high-throughput sequencing. The core part of the primer is composed of 4 stable and unchanged bases. In addition, to increase the flexibility and adaptability of the primer, the design also includes 3 variable bases (the combination of NWN and NYN), forming the so-called elastic sequence. This design enables the primer to adapt and bind to a variety of different sequences in the genome, thereby increasing the breadth and depth of sequencing. These primers can bind to the introns, coding sequences, promoter upstream 2000 bp, and DNA sequences in non-gene regions of the genome. By using the seqtk tool to randomly screen sequencing data and format conversion. Then, using the BLAST tool to quickly compare the converted data with the sequences in the NCBI's NT database to identify potential matching regions, and then calculate the similarity scores of these regions, returning the highest-scoring matching sequence. Statistically identify the species to which the matching sequence belongs, and obtain the identification conclusion of the sample through the calculation formula and rules in this document.

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