An efficient and cost-effective method for primer-induced nucleotide labeling for massive sequencing on next-generation sequencing platforms

Junjie Guo,Jie Zeng,Tao Cheng,Yide Li,Han Xu

doi:10.1038/s41598-019-38996-8

Abstract

Next generation sequencing (NGS) technologies play a powerful role in the preparation of large DNA databases such as DNA barcoding since it can produce a large number of sequence reads. Here we demonstrate a primer-induced sample labeling method aiming at sequencing a large number of samples simultaneously on NGS platforms. The strategy is to label samples with a unique oligo attached to the 5′-ends of primers. As a case study, 894 unique pentanucleotide oligoes were attached to the 5′-ends of three pairs of primers (for amplifying ITS, matK and rbcL) to label 894 samples. All PCR products of three barcodes of 894 samples were mixed together and sequenced on a high throughput sequencing platform. The results showed that 87.02%, 89.15% and 95.53% of the samples were successfully sequenced for rbcL, matK and ITS, respectively. The mean ratio of label mismatches for the three barcodes was 5.68%, and a sequencing depth of 30 ×to 40× was enough to obtain reliable sequences. It is flexible to label any number of samples simply by adjusting the length of oligoes. This easy, reliable and cost efficient method is useful in sequencing a large number of samples for construction of reference libraries for DNA barcoding, population biology and community phylogenetics.

Highlights

Since the DNA barcoding concept was proposed by Hebert et al.[1]
No significant differences (p ≥ 0.05) in PCR success percentage were observed between both types of primers for each barcode, while significant differences exist among the three barcodes (p < 0.05)
The primers linked with pentanucleotide labels showed a good performance in the PCR and pyrosequencing process in the present study

Summary

Introduction

Since the DNA barcoding concept was proposed by Hebert et al.[1]. DNA-based taxonomic identification has become a very important tool, and great progress has been made on the improvement. Methods, with unique labels or tags linked to each sample in order to distinguish them[25,26,27,28,29] These attempts made accurate identification of both gene fragments and samples, and made NGS more scalable and efficient, there were still some drawbacks. One such a drawback is that only a small number of samples (less than 300) can be identified at once using two-nucleotide tags in some cases, and cannot meet the demand for massive sequencing. We describe how the plant DNA barcodes of ITS, rbcL and matK of 894 pant samples were sequenced simultaneously on the Roche 454 plus platform This method is applicable on any other NGS platforms

Methods

Results

Conclusion