MetaFast protocol for PCR amplicon analysis of metagenomic DNA

MetaFast is a simple and cost-effective method to answer complicated metagenomic questions with high precision using a PCR approach.

The MetaFast protocol has been developed by Fasteris for Dr. Pierre TABERLET (CNRS and University Grenoble Alpes, Grenoble, France) to analyse metagenomic amplicons with both low diversity and low complexity. The protocol is based on metabarcoding of the amplicons from specific regions (e.g. 16S / 18S / ITS or others).

It allows pooling several PCR products before library preparation step, hereby reducing significantly the cost of the library preparation.

The initial step is a single PCR amplification using barcoded primer pairs. Thsi step can be done at your site (in your laboratory) or at Fasteris (see also the list of standard Fasteris primers in the table below). Alternative custom primers can be used upon request and when respecting the MetaFast details and specifications.

The PCR-free library preparation step, combined with the Fasteris internal protocol optimization, helps to overcome chimera effect of PCR and hereby improving the overall sequencing quality.

Requirements:  100 ng of gDNA in 25 µL water or
                           1 µg of equimolar amplicon pool in 30 µL water.

NB: Please note that due to amplicon reduced diversity we recommend using specific barcode construction or increase of the amount of PhiX in the sequencing run.

NB: A minimum of 100’000 paired-reads with 50-100 bp overlapping region per amplicon, i.e. with insert size of max 450 bp, is recommended for statistical analysis.

NB: In case if the initial PCR is performed at Fasteris, the final data will be provided as “barcode-sorted, trimmed and overlapped reads” with one FastQ file per sample (if applicable).The additional treatments aim to facilitate for our clients the downstream bioinformatics analysis. For customer-made amplicons (PCR products) this bioinformatics step is available and will be performed only on request.

 

Please also note: Due to the diverse nature of metagenomic samples (various species, variable target genes, variable target regions, fluctuating GC content, etc.) Fasteris cannot guarantee for any minimal percentage of overlapping reads that finally will be provided.

Target Forward primer Reverse primer Fragment size Reference
16S*

341F* (V3 region)

CCTACGGGNGGCW GCAG

805R* (V4 region)

GACTACHVGGGTATC TAATCC

~ 550 bp Klindworth et al. 2012
ITS2

ITS3_KYO2

GATGAAGAACGYAG YRAA

ITS4

TCCTCCGCTTATTGAT ATGC

300-400 bp Toju et al. 2012
18S

18S_0067a_deg

AAGCCATGCATGYCT AAGTATMA

NSR399

TCTCAGGCTCCYTCTC CGG

~ 350 bp Dollive et al. 2012
(*) 16S broad range primers, 341F binds to bacteria, 805 R matching both bacteria and some archea.

 

Available NGS sequencing options:

By our routines, Fasteris offers sequencing on Illumina NextSeq 500 and MiSeq platforms. The output and coverage yielded for the PCR analysis can be adjusted to your needs. The above given PCR products should preferentially be sequenced applying 2x300 bp (possible only on MiSeq) or 2x250 bp paired-reads (possible on MiSeq, MiSeq Nano and NovaSeq 6000).

Bioinformatics analysis:

For bioinformatics analyses such as Species Diversity Comparison please visit our bioinformatics service webpage.

Last updated: 2020/01/14