16s rdna sequence analysis software#
A phylogenetic tree was constructed for each sample based on the neighbor-joining method using the MEGA software package version 6 14 and the evolutionary distance was calculated based on the Jukes & Cantormodel 15. After obtaining the alignment results for each sample’s sequence, the information for 30-60 phylogenetic neighbors was downloaded for phylogenetic analysis. The top 30 sequences with the highest scores were selected for the calculation of pairwise sequence similarity using a global alignment algorithm 13, which was implemented on the EzTaxon server ( 11). The identification of phylogenetic neighbors was initially carried out by the BLASTN 12 program against the EzTaxon database. EzTaxon is a public database containing type strains with valid published prokaryotic names and representative sequences from uncultured phylotypes 11.
16s rdna sequence analysis full#
The sequences for both the first 500 bp (5F to 531R) and the nearly full gene sequences (5F to 1492R) were compared against the EzTaxon database. Study ApproachĪ total of 208 diverse “in house” sequences covering 131 genera were randomly selected for further analysis. The purpose of this study was to examine the hypothesis that the first 500 bp of the 16S rRNA gene is sufficient for identification of bacteria recovered during the execution of EM programs. Use of the entire 1500 bp versus the first 500 bp 16S rRNA gene sequence in bacterial identification has attracted interest from the biopharmaceutical industry, but the number of studies and the data supporting this option are limited. Traditionally, sequence-based identifications made on samples from EM programs have been made based on the fi rst 500 bp of the 16S rRNA gene. The first 500 bp may even show increased resolution between certain species since this region can show slightly more diversity than the remaining sequence 4. On the other hand, Clarridge (2004)4 claimed that for most clinical bacterial isolates, evaluating the first 500 bp is sufficient for identification. For the identification of bacteria, it is reported that the full length 16S rRNA gene sequence analysis may provide better resolution for certain species 4,9,10. For the publication and taxonomic classification for novel bacterial species, the full length of the 16S rRNA gene sequence followed by phylogenetic relationship analysis with other closely related species is necessary 8. The entire 16S rRNA gene sequence is approximately 1500 base pairs (bp) and consists of highly conserved regions which provide a broad taxonomic spectrum, and nine hypervariable regions (V1 – V9) that allow high taxonomic level discrimination 6,7. There are several different options available for bacterial identification however, the use of 16S rRNA gene sequences has been considered the most powerful and accurate tool, while conventional phenotypic methods often show major weaknesses 2-5. The EM program is only effective if the organisms recovered from the facility are accurately identified, so the information gathered can be used to understand the microbial control through tracking and trending and dictate appropriate remediation activities. The Food and Drug Administration (FDA) has published guidelines for the production of sterile drugs by aseptic processing which includes a section on EM programs, and the USP general information chapter “Microbiological Control and Monitoring of Aseptic Processing Environments” also contains detailed information regarding EM programs 1. A properly executed EM program provides an early warning of potential contamination problems due to equipment failure, inadequate cleaning, or deficiencies in staff hygiene training, for example, so that problems can be corrected to prevent adulteration of the end product. The EM program is a biological surveillance system which enables companies to quickly identify organisms which are transient or resident in their facilities before these organisms have an opportunity to contaminate a product. Environmental Monitoring (EM) programs are the cornerstone of understanding the microbial ecology in a manufacturing facility and have become a regulatory requirement for most manufacturers. Bacterial Identification in the biopharmaceutical industry, especially in manufacturing facilities, is very important because an occurrence of a problematic microorganism in the final product could be harmful for the end user and detrimental to a company’s finances and reputation.
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