An efficient and cost-effective method for primer-induced nucleotide labeling for massive sequencing on next-generation sequencing platforms
Autor: | Han Xu, Junjie Guo, Yide Li, Tao Cheng, Jie Zeng |
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Jazyk: | angličtina |
Rok vydání: | 2019 |
Předmět: |
0301 basic medicine
DNA Plant Computer science Plant genetics Pcr cloning lcsh:Medicine Computational biology DNA barcoding Article DNA sequencing Deep sequencing 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Phylogenetics DNA Barcoding Taxonomic Nucleotide lcsh:Science DNA Primers chemistry.chemical_classification Multidisciplinary Nucleotides lcsh:R High-Throughput Nucleotide Sequencing Plants 030104 developmental biology chemistry lcsh:Q Primer (molecular biology) 030217 neurology & neurosurgery DNA |
Zdroj: | Scientific Reports, Vol 9, Iss 1, Pp 1-7 (2019) Scientific Reports |
ISSN: | 2045-2322 |
Popis: | 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. |
Databáze: | OpenAIRE |
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