BS ISO 23732:2021 pdf download – Marine technology — Marine environment impact assessment (MEIA) — General protocol for observation of meiofaunal community
7 Procedures for metagenomic analysis (molecular analysis) 7.1 General For metagenomic analysis, DNA or RNA shall be targeted. When RNA is used, it can be easily matched with the results of imaging flow cytometry. RNA is subjected to reverse transcription reaction and treated as cDNA (complementary DNA) (see 7.4.2). Examples of metagenomic analysis are shown in Annex B.
7.2 Nucleic acids (DNA/RNA) extraction A DNA/RNA extraction kit by using beads beating method for soil sample is recommended. The same DNA/RNA extraction method for the sample collected at the same sampling point during time course observation should be used. For RNA extraction, purification with DNase should be performed to digest all DNA fragments.
7.3 PCR primers For metagenomic analysis by NGS, the target gene that is used for taxonomic identification is recommended [e.g. 18S rRNA, ITS (internal transcribed spacer), 28S rRNA (large subunit rRNA), and mitochondrial COI (cytochrome oxidase subunit I)]. The target gene should be chosen depending on the purpose. The primers for this step, the sequence should consist of specific sequence for target gene sequence and overhang adaptor sequence for NGS.
7.4 PCR protocol
7.4.1 DNA In PCR (amplicon PCR), DNA fragments are amplified by repeated cycles (thermal cycling), which consists of 3 steps, (1) denaturation (denaturing a double stranded DNA into two single stranded DNA molecules by heat), (2) annealing (annealing of a primer with a single stranded complementary DNA), and (3) elongation (elongation of a single stranded DNA from the site where a primer annealed). PCR conditions (number of cycles and reaction temperature) shall be designated for appropriate amplification. The amplifications should be done in 3 replicate PCR reactions for each sample, after electrophoresis to check the amplicon, combined triplicate PCR reactions of the same sample into a single volume.
7.4.2 RNA (cDNA library production) A reverse transcription shall be performed for preparing a cDNA library using the extracted RNA. Usually, reverse primer is used to extend the target region. Only one reaction should be done for a single primer extension. The generated cDNA library is used as templates for amplicon PCR (see 7.4.1). At the same time, to confirm extracted RNA quality (to check the DNA contamination), the same PCR should be performed by RNA before reverse transcription. If the amplified fragments are seen on the agarose gel as a band by RNA template PCR, the RNA may contain co-extracted DNA fragments. In that case, it should purify the RNA.
7.5 Check and purification of PCR amplicon
The amplicons should be checked by agarose gel electrophoresis. If the DNA fragment is amplified with appropriate PCR conditions, the expected length amplicon is seen as a band on the agarose gel. After checking the amplicons, 3 replicate PCR reactions of the same sample should be combined into the single volume, and proceed to the purification step.
Amplicons shall be purified using magnetic beads to remove residual PCR primers and reagents, according to the supplier’s manual. Magnetic beads can purify the large DNA fragments (such as amplicons) without excess primers, non-specific short fragments, salts, and enzymes by paramagnetic bead technology.
7.6 Index PCR Performance of NGS depends on the type of a sequencer; however, it is known to read 10 million sequences (called reads) in one analysis. So, it is possible to analyse multiple samples simultaneously and be often used to sequence all of mixed multiple amplicons. Therefore, to distinguish the amplicon which derived from different source, tag sequence is added to each amplicon by index PCR. Primers for index PCR shall be comprised overhang adaptor sequence which is the same sequence of amplicon PCR, tag sequence (index sequence), and sequence adaptor for NGS.