1. Culturable Bacterial Communities Analyzer DIANA VANESSA SARRIA-ZUNIGA ELIANA TORRES-ZELADA April 29, 2016

2. Bacterial 16S rRNA Modified from: http://www.alimetrics.net/en/index.php/dna- sequence-analysis PCR amplification of region V3-V6 Modified from: http://microbiollogy.blogspot.com/2014_08_01_archive.html 2. Sanger sequencing PCR product V3-V6

3. REQUIREMENTS: 1.Access to Purdue servers 2.Access to Purdue Bioinformatics Core facility: -module perl/5.20.1 -module CAP3/12.21.07 -module muscle/3.8.425 -module mothur/1.34.4 -module usearch/5.2.236 3. GAST ( Global Assignment of Sequence Taxonomy for SSU rRNA ) files

4. 1. DOWNLOAD: TRIMMED SEQUENCES AND QUALITY SCORE FILES Trimmed sequences Quality score file LWP::UserAgent Screen scrapping  links

5. 2. ASSEMBLY CAP3 FORWARD AND REVERSE COMPLEMENT SEQUENCES  CONTIGS Perl script: Reverse complement One file per sample  Run CAP3 per sample  Store all contigs in one file

6. 3. ALIGNMENT MUSCLE: ALIGN CONTIGS TO SEARCH REGION V3, V6 AND V3-V6 Perl Script: search primers V3F, V3R, V6F, V6R and extract the three regions Input: contigs  MUSCLE  Output: Alignment of contigs

7. 4. TAXONOMY ASSIGNMENT GAST File with all regions V3 File with all regions V6 OUTPUT GAST

8. 6. DATABASE MySQL One table i. Colony ID ii. Taxonomy GAST Region V3: Domain;Phylum;Class;Order;Family;Genus;Species iii. Taxonomy GAST Region V6 Domain;Phylum;Class;Order;Family;Genus;Species. iv. BLAST Region V3-V6 v. Final Taxonomy 5. FINAL IDENTIFICATION Perl script: compare taxonomy assigned for V3 and V6 region of each colony. If they agree  final identification. If they do not agree  submit the V3-V6 fasta file to BLAST and keep the best 5 hits.

9. THANK YOU QUESTIO NS??