A Practical Method for Barcoding and Size-Trimming PCR Templates for Amplicon Sequencing

Abstract

Sample barcoding facilitates the analysis of tens or even hundreds of samples in a single next-generation sequencing (NGS) run, but more efficient methods are needed for high-throughput barcoding and size-trimming of long PCR products. Here we present a two-step PCR approach for barcoding followed by pool shearing, adapter ligation, and 5' end selection for trimming sets of DNA templates of any size. Our new trimming method offers clear benefits for phylogenetic studies, since targeting exactly the same region maximizes the alignment and enables the use of operational taxonomic unit (OTU)-based algorithms.

Microbial community composition but not diversity changes along succession in arctic sand dunes

“…We used M13 system for library preparation as described by Mäki et al . (). Prior to sequencing, the 16S rRNA gene amplicons were size fractionated (Pippin Prep, Sage Science) to eliminate the mitochondrial amplicons.…”

Plants Assemble Species Specific Bacterial Communities from Common Core Taxa in Three Arcto-Alpine Climate Zones

“…The nested primers targeting the V6-V8 regions of 16s rRNA gene enable elimination of plant chloroplast 16S rRNA gene amplicons as well as separation of endophyte amplicons from plant mitochondrial amplicons by size fractionation (799f–1492r, Chelius and Triplett 2001) and produce an amplicon with high phylogenetic coverage and optimal size for IonTorrent sequencing (1062f–1390r). Primers 1062f (Ghyselinck et al, 2013) and 1390r (Zheng et al, 1996) were tagged with M13 sequences to enable sample barcoding as described below and in Mäki et al (2016). Both reactions had 1 μl of sample DNA, 1x PCR buffer, 1 mg/ml of BSA, 0.2 mM dNTP's, 0.3 μM of each primer and 1250 U/ml GoTaq DNA Polymerase (Promega, WI, USA) in a 30 μl reaction volume.…”

“…Sequence libraries were prepared by running a third PCR to attach the M-13 barcode system developed by Mäki et al (2016). Amplicons from second PCR were diluted 1:5 and re-amplified using barcode attached M13 system as forward primer and 1390r-P1 with adaptor A as a reverse primer.…”

Resistant ammonia‐oxidizing archaea endure, but adapting ammonia‐oxidizing bacteria thrive in boreal lake sediments receiving nutrient‐rich effluents

“…The PCR products were purified, DNA concentration measured, and the samples pooled in equal DNA amounts as described for 16S rRNA genes. In order to adjust the length of the archaeal and bacterial gene amplicons (~ 600 bp) for the Ion Torrent™ sequencing (recommended amplicon length ~ 400 bp), the sample pool was sheared, ligated to P1 adapter, size‐selected and re‐amplified as a single reaction as described in the study by Mäki and colleagues (). The sequencing protocol downstream followed that which was described for the 16S rRNA gene sequencing.…”

“…Thermal cycling consisted of 10 min initial denaturation at 95 C, followed by 24 cycles of 95 C for 30 s, 50 C for 30 s and 72 C for 60 s, followed by final elongation at 72 C for 10 min, was conducted on Bio-Rad CFX96 Real-Time System (Bio-Rad Laboratories). About 2 μl of the product was used as a template in a second PCR where Ion Torrent PGM sequencing adapters and barcodes were added to the ends using linker and fusion primers (0.04 μM of M13_515FB, 0.4 μM of IonA_IonXpressBarcode_M13 and P1_806R) in eight additional cycles with conditions otherwise identical to the first amplification (Mäki et al, 2016). Products were purified with Agencourt AMPure XP purification system (Beckman Coulter Life Sciences, Indianapolis, IN, USA), quantified as earlier described, and pooled in equimolar quantities for sequencing on Ion Torrent PGM using Ion PGM Hi-Q View OT2 Kit for emulsion PCR, PGM Hi-Q View Sequencing Kit for the sequencing reaction and Ion 316 Chip v2 (all Life Sciences, Thermo Fisher Scientific).…”