Principles and processes for high-throughput sequencing of amplificationor sequencing, such as 16S/18S/ITS.

In agriculture, forestry, animal husbandry, breeding, medicine and many other industries, microbiodiversity is one of the more hot research projects in recent years.
as the most commonly used detection technology of microbiodiversity, "second-generation high-throughput sequencing technology", because of its more detection data, analysis content, with its price is getting lower and lower, is now accepted and applied by many teachers.
here is a brief introduction to the principles and processes of high-throughput sequencing of amplification or sequencing, such as 16S/18S/ITS.
the current market for more second-generation sequencing platform sillumina company's Miseq, Hiseq platform.
Illumina sequencing uses edge synthetic edge sequencing (Sequencing by synthesis, SBS).
because of its sequencing length, data output and so on, but also subdivided into 2 x 300, 2 x 250, 2 x 150 and other models.
, before introducing the inspection process, introduces the chip (flowcell), an important component of the illumina sequencer.
there is a thin slice called flowcell in the Illumina sequencer, there are multiple lanes on the flowcell, there are many oligo "joints" on the flowcell, and the purpose sequence of the samples being tested will be "enriched" by the bridge PCR during the later sequencing process.
flowcell is an important structure in the sequencer, where both synthesis and sequencing reactions are performed.
the number of flowcells and the number of lanes that can be placed are different
the sequencer model.
16S/18S/ITS and other amplifier sequencing is mainly through the detection of a specific gene to obtain a certain type of microbiodiversity of bacteria / eukaryotic microorganisms / fungi in the sample, the detection method generally includes DNA extraction, library construction, machine sequencing, bio-information analysis four steps.
sample DNA extraction to the sample, the corresponding DNA kit to the total DNA extraction of the sample, different samples using different DNA extraction kit.
such as stool-specific feces-specific extraction kits (Stool DNA Kit), soil-based soil DNA extraction kits (Soil isolation DNA Kit).
the DNA quality control of extraction is qualified and then enters the library to build.
library to build 16S/18S/ITS and other amplifier sequencing is the target gene fragment of detection, not the heavy DNA in the sample, so it is necessary to use specific primers to amplify the target fragment, and then add the connector stonas(index), sequencing primer, and "index" required by the sequencer to the sides of the target fragment to build the library.
-specific primers are important for sequencing results.
the same sample is amplified with different specific primers, and the results are often different.
for the current 16S rDNA has 9 conservative and variable zones, since the current sequencing instruments can mostly measure 400-500bp, so it is recommended to choose a dual V zone, the current literature is commonly used in the V3-V4 zone, V4-V5 zone.
additional barcode is added to distinguish samples, currently in sequencing often hundreds of samples at the same time, without adding the barcode, you can not know which samples are. After the
is prepared on the machine sequencing library, the library is sequenced by the upper level.
on the machine, one end of the DNA fragment in the library and the Oligo connector on the surface of the flowcell are attached to the DNA fragment saued on the flowcell in a complementary way.
on the flowcell, a series of bridge-type PCR amplifications generates a DNA cluster, where a dna fragment can be amplified to a cluster.
added modified DNA polymerases and fluorescently labeled dNTP to detect dNTP signals bound to DNA fragments to obtain base information.
synthesizes only one base per cycle.
count the fluorescent signals collected in each round to learn the sequence of DNA fragments.
the original sequence data of the sample after the sequence of bioinformatics is completed.
facing the massive ATCG data, can not be used directly, need to use professional software to make it easy to understand and visual.
commonly used software are mothur, QIIME, R language and so on.
optimize the original sequence by mothur or QIIME software, remove the no-bar sequence, include N base, low quality, chimeric, etc., use the optimization sequence to cluster the OTU, and use the OTU to evaluate the sequence and the database.
subsequent visual and statistical analysis are based on OTU tables or physical tables.
current routine analysis includes the type and relative abundance of microorganisms in the sample, the alpha diversity index (shannon, chao, simpson, etc.), the analysis of beta diversity (PCA, PCoA, heatmap, etc.) showing differences between samples, and the differences between groups (Lefse analysis, ANOVA analysis, etc.).
Source: A ChengBIO Pony Research Tribe.