An improved method for bacterial identification through 16S rDNA sequencing using double set of primers: a case study.

Amanat  Ali; Sohail  Hameed; Ivan J.  Oresnik; Muhammad  Shahid; Mohsin  Tariq; Naveed  Ahmad

Amanat Ali National Institute for Biotechnology and Genetic Engineering (NIBGE), Jhang Road, Faisalabad, Punjab, Pakistan;Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada;Soil Science Division, Nuclear Institute of Agriculture (NIA), Tandojam, Sindh, Pakistan

Sohail Hameed National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan

Ivan J. Oresnik Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada

Muhammad Shahid National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Punjab, Pakistan;Department of Bioinformatics and Biotechnology, Government College University, Faisalabad, Pakistan

Mohsin Tariq National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Punjab, Pakistan;Department of Bioinformatics and Biotechnology, Government College University, Faisalabad, Pakistan

Naveed Ahmad National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan

16S rDNA sequencing is an established technique for molecular identification and phylogenetic studies of bacteria. In most of the previous studies, single set of primers (16S forward and 16S reverse) was unlikely to amplify and sequence full fragment of 16S gene and the product size remained shorter. Thus, an improved method was adopted to sequence the fulllength 16S rDNA fragment employing an additional set of internal primers (Henk 16S and Hoff 16S) along with external primers (Loff 16SF and Loff 16SR) in a single run to enhance the precision in bacterial identification by getting a sequence length of over 1300 bp. Moreover, we presented another way to tackle the missing sequences and some faulty readings in the raw sequence data that appear to be another impediment causing imprecise identification of bacteria. Therefore, peaks crossmatching was done while editing the raw sequence data prior to alignment through NCBI BLAST. Finally, CAP3 sequence assembly online program was employed for precise assembling of the sequence data obtained from each of the four primers to obtain a single contiguous sequence for greater precision in bacterial identification. This approach will certainly help taxonomists to describe novel bacterial species.