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

Scientific Reports
Junjie GuoJie Zeng

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 ...Continue Reading

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Citations

Aug 14, 2020·PloS One·Guobin Chen, Xianzhong Xie

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Methods Mentioned

BETA
PCR
454 sequencing
PCRs
electrophoresis

Software Mentioned

BLAST
NGSQCtoolkit
Sequencer
TNAssembler

Related Concepts

Related Feeds

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

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