Workshop on the analysis of microbial sequence data using ARB August 13 - 14, 2009 University of Hawaii Instructors: Amy Apprill, UH & WHOI (apprill@whoi.edu) Megan Huggett, UH (huggett@hawaii.edu) Sponsored by:

By the end of the workshop you will be able to: Efficiently work with raw nucleotide sequence data and prepare sequences for upstream analysis Be familiar with the ARB software package Align sequences using ARB Construct phylogenetic trees using ARB Utilize a few of the web-based resources available for sequence analysis & phylogenetics Design probes & primers using ARB Construct a functional gene database using ARB

Workshop schedule Thursday, August 13: 9:00 – 10:00am: Overview of workshop, Lecture: Introduction to the theory of phylogenetic analyses and the ARB program 10:00 – 10:40am: Tutorial 1 – Clean-up, assemblage and quality control of sequence data 10:40 – 11:00am: Coffee break 11:00 – 11:15am: Lecture: Sequence Alignments 11:15 – 12:30pm: Tutorial 2 - Importing and aligning sequences into an ARB database 12:30 – 1:30pm: Lunch break – Discussion: Student introductions 1:30 – 2:00pm: Free time to complete tutorial 2 2:00 – 2:30pm: Lecture: Overview of phylogenetic comparisons and tree-building 2:30 – 3:40pm: Tutorial 3 – Creating a phylogenetic comparison of the newly imported and aligned sequences 3:40 – 4:00pm: Afternoon break 4:00 – 4:30pm: Tutorial 4 – Creating a sequence alignment filter 4:30 – 5:00pm: Tutorial 5 – Using BLAST to identify related sequences

Workshop schedule Friday, August 14: 9:00 – 9:30am: Recap of day 1, intro to assignment; question & answer session  9:30 – 10:40am: Free time to complete tutorials & assignment 10:40 – 11:00am: Coffee break 11:00 – 12:00pm: Free time to complete assignment 12:00 – 1:00pm: Lunch break – Discussion: Opportunities in science education for PhD’s  1:00 – 2:00pm: Free time to complete assignment, question and answer session  2:00 – 2:30pm: Lecture: Primer and probe design  2:30 – 3:40pm: Tutorial 6: Primer and probe design  3:40 – 4:00pm: Afternoon break  4:00 – 5:00pm: Tutorial 7: Creating and working with new databases

Lecture 1: Introduction to the theory of microbial phylogenetics & the ARB program

Phylogeny in microbiology Morphology Requires cultured isolates (e.g. Petri dishes) Limited information content Chemical cell components (phospholipids, fatty acids) Usually misses 99% of diversity because: Culture conditions can’t be copied Many species can’t be exist in isolation

Phylogeny in microbiology The alternative Using molecular tools to uncover previously unknown microbial diversity 16S/18S rRNA has become the standard molecular marker Of course, we still need isolates! Results from microbial genome and metagenome sequencing can particularly guide isolation studies Sequence of molecular marker genes - High information content, low cost, limited labor

What makes a good phylogenetic marker gene? Presence in all organisms Functional constancy Complexity Size Conserved and variable structural elements Comprehensive database Some examples: 16S rRNA, 23S rRNA, ATP synthase, RNA polymerase….

Ribosomal RNA as a phylogenetic marker  Small subunit (SSU) or 16S ribosomal RNA gene 16s rRNA gene Genomic DNA Transcription Folding rRNA Becomes part of the ribosome, vital for protein synthesis

Ribosomal RNA as a phylogenetic marker Advantages: Ubiquitous distribution Regions of high variability Constant rate of evolution Naturally replicated in situ Conserved & variable regions Large databases available Conserved PCR primer sets for genes

Ribosomal RNA as a phylogenetic marker Disadvantages: Many copies per genome (microheterogeneity) Limited information (only 1500bp, relatively few of which are informative) Different species have identical 16S rRNAs

Secondary structure of ribosomal RNA: constrained mutation across 2-3 billion years Escherichia (Bacteria) Methanococcus (Archaea) Saccharomyces (Eukarya)

Secondary structure of ribosomal RNA Complex pattern of short double-stranded stems, unpaired single-stranded loops and bubbles

Secondary structure of ribosomal RNA Eucarya Bacteria Archea Conserved

Modern tree of life based on 16S/18S rRNA sequences

Methods to analyze rRNA Environmental sample DNA extraction Detection of cells PCR amplification Fluorescence in situ hybridization Cloning & sequencing Probe design Sequence analysis Sequence database

Phylogenetic sequence analysis - the bad news Phylogenetic analysis needs: Good sequence data Proper alignment, analysis and statistical testing Tidy presentation “Probes are only as good as the data behind them” Phil Hugenholtz (1988) Good data are critical

Phylogenetic sequence analysis - the good news Arb can help you: Organize and collect your data Align your data Make trees Design probes

Phylogenetic sequence analysis steps Sequence determination Preparation of raw sequence data (removal of cloning vector, clean ends, quality control, assemble contigs) Import sequences into database Alignment of sequences to secondary structure Phylogenetic comparison

Gene cloning & sequencing TATGCTTGCAA TTGGTTCT ACGCTGCT ATTGCCTA DNA PCR amplify gene Clone DNA using E. Coli & plasmid insert ATTGCCTA TTGGTTCT TATTGCCTT TTTGCCTT ATTGCCTT Sequence

Cloning vector

Cloning vector

Sequence analysis software – Sequencher (Mac or PC)

SSU rRNA gene databases Silva rRNA based database project (www.arb-silva.de) Well maintained SSU & LSU databases available Most recent SSU database contains 368,368 quality filtered sequences! Living Tree Project databases (typed species) Sequence alignment function (SINA) Green genes (www.greengenes.lbl.gov) SSU rRNA gene database Sequence trimming functions Alignment functions Probe & primer design functions

ARB Tutorials Importing sequences into ARB Aligning sequences using ARB & SINA web-based aligner Adding sequences to ARB database parsimony tree Creating sequence filters Importing sequences from GenBank into ARB Constructing a phylogeny of sequences Probe & primer design Constructing databases for functional genes

Tutorial 1 objectives Become familiar with sequence chromatograms Trim cloning vector sequence files Trim the ends of sequences Assemble sequence fragments into contiguous sequences Export the sequences into a format for ARB