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A Two-Step PCR Protocol Enabling Flexible Primer Choice and High Sequencing Yield for Illumina MiSeq Meta-Barcoding

Journal Article · · Agronomy (Online)
 [1];  [2];  [2];  [3]
  1. University of Florida, Quincy, FL (United States); Academia Sinica, Taipei (Taiwan)
  2. Michigan State University, East Lansing, MI (United States)
  3. University of Florida, Quincy, FL (United States)
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High-throughput amplicon sequencing that primarily targets the 16S ribosomal DNA (rDNA) (for bacteria and archaea) and the Internal Transcribed Spacer rDNA (for fungi) have facilitated microbial community discovery across diverse environments. A three-step PCR that utilizes flexible primer choices to construct the library for Illumina amplicon sequencing has been applied to several studies in forest and agricultural systems. The three-step PCR protocol, while producing high-quality reads, often yields a large number (up to 46%) of reads that are unable to be assigned to a specific sample according to its barcode. Here, we improve this technique through an optimized two-step PCR protocol. We tested and compared the improved two-step PCR meta-barcoding protocol against the three-step PCR protocol using four different primer pairs (fungal ITS: ITS1F-ITS2 and ITS1F-ITS4, and bacterial 16S: 515F-806R and 341F-806R). We demonstrate that the sequence quantity and recovery rate were significantly improved with the two-step PCR approach (fourfold more read counts per sample; determined reads ≈90% per run) while retaining high read quality (Q30 > 80%). Given that synthetic barcodes are incorporated independently from any specific primers, this two-step PCR protocol can be broadly adapted to different genomic regions and organisms of scientific interest.
Research Organization:
Chicago Office, Argonne, IL (United States)
Sponsoring Organization:
Ministry of Science and Technology; National Science Foundation (NSF); U.S. Department of Agriculture
Grant/Contract Number:
SC0020403
OSTI ID:
2570226
Journal Information:
Agronomy (Online), Journal Name: Agronomy (Online) Journal Issue: 7 Vol. 11; ISSN 2073-4395
Publisher:
MDPICopyright Statement
Country of Publication:
United States
Language:
English

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Figures / Tables (10)

Table 2(p. 4)table Table 2
Figure 3(p. 6)figure Figure 3
Table 3(p. 8)table Table 3
Figure 6(p. 15)figure Figure 6
Figure 2(p. 5)figure Figure 2
Figure 2(p. 5)figure Figure 2
Figure 5(p. 13)figure Figure 5
Figure 1(p. 3)figure Figure 1
Table 1(p. 4)table Table 1
Figure 4(p. 12)figure Figure 4

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Related Subjects

16S rDNA
ITS
Illumina
MiSeq
amplicon sequencing
metabarcoding