Q-PCR Analysis of AOM at the Mariana Forearc Mud Volcanoes: Primer design for mcrA

1. Q-PCR Analysis of AOM at the Mariana Forearc Mud Volcanoes: Primer design for mcrA Kelsey Jesser Alex Wipf Denise Castro Josh Gross

2. Kelsey’s thesis… • Extract genomic DNA • Run Q-PCR analysis on genes associated with anaerobic oxidation of methane (AOM) • Looking for organisms that metabolize methane! http://www.meltonengineering.com/Breaking%20News.htm Adapcted from Curtis et. al submitted

3. Wanted: Awesome Q-PCR Primers*,** • *Must amplify only the gene of interest (mcrA) • **Cannot be biased for any particular type of organism

4. Plan of Attackprimer design for mcrA • Multiple sequence alignment (MSA) of mcrAsequences to find conserved regions of DNA • As many sequences as we can get! • Design primers based on MSA • Test primers against complete Archaeal genomes from the UCSC Archaeal Genome Browser

5. Methods: MSA and primer design • Biology: Provide reference mcrAsequence for BLAST search of NCBI database • CS: Pull lots of mcrAsequences from NCBI using Biopython scripts, align sequences in ClustalW • Biology: Use alignment to design unique primers in Primer3, look at primers in IDT Oligo Analyzer

6. Pulling Sequences • Gene Sequences • Qblast API • NCBIWWW.qblast(“blastn”, “nt”, mcrA, hitlist_size = 200) • Fasta format

7. Multiple sequence Alignment

8. Primer Design

9. Methods: Genome Walking • CS: Pull 120 complete genome sequences from UCSC Archaeal Genome Browser • Biology: Provide published reference primer set (Hallam et. all 2003), design unique primer sets • CS: Create algorithm to “walk” candidate primers along complete genomes to check primer specificity • *want primers that will amplify ONLY the mcrA sequence of interest across the greatest number of Archaeal genomes

10. Pulling Sequences • Whole Genome Sequences • 120 species of archaea. http://archaea.ucsc.edu/genomes/archaea/

11. Pulling Sequences

12. Results • We tested primers on species of Archaea • Primers we designed • success rate of 2/120 for the left and 4/120 for the right • Primers from publication • 0/120 and 9/120 respectively • Tried to fix by trying all 20-length seqs in conserved regions on all 120 species • 34 left, best match rate was 2 • 111 right, best match rate was 6 • This run took nearly 7 hours • Nearly all matches were Methano- species

13. Troubleshooting • Downloading correct sequence data • Sequence sources • Assumptions • Data matching • We assumed our MCRA data (what we aligned) was a good sampling of the sequenced species of Archaea (what we tested against). • MCRA might not be as conserved as we originally believed – all we were aligning were Methano- • False negatives?

14. Reflections • We learned LOTS! • In hindsight… • Start earlier • Contingency plans • May have oversimplified the problem

15. Future Work • Keep working on primer design and genome walking • Use this method to design Q-PCR primers for other AOM functional genes • Apply genome walking algorithm to bacterial genomes • One gene of interest (dsrA) is actually a bacterial gene