The process of 16s rRNA sequencing (original)

1.First step is the extraction of genomic DNA.

It will has an extraction of DNA in the genome of samples.after the completion of DNA extration,we will conduct a detection on the extracted genomic DNA via 1% Agarose gel electrophoresis.

2.PCR augmentation

According to the specified sequencing area(the 16s region on rna sequence),we can design and synthesis the primer with special barcode,which can distinguish and augment these sequences when conducting a PCR process.

3.The depuration of DNA sequence output through PCR.

The aim of the step is to delete the short size sequence ,so that the quality of DNA segment above 300bp can be selected and use for the following operation.

4.The quantitative analysis

The PCR products were quantified by the quantification system,followed by the corresponding proportions of the samples required by each samples,we will have a homogeneous anlysis.

5.In this step we will use the Illumina PE 250 to make a sequencing on DNA segments.And the Illumina PE250 sequencing will get so many of PE reads.

6.Reads overlap

The PE reads contents about two sequences which is the read1 and read2.According to the overlap between the read1 and the read2 we can make a mergence,simultaneously,we will do the quality control,split up and filtration.

7.we will have a differentiation based on each samples and then conduct a cluster analysis of OTUs ,and taxonomic analysis of species via blasting to the Database(GREEGEN).

8.we can get an OTU abundance table,which can make a lot of enchanting plots.