Collecting and Storing Sequences In the laboratory Heather Helm UPR Sequencing Facilities Manager

Topics for Today The Cycle Sequencing Reaction Reaction Overview Interferences Sequencing Analyzers Slab Gel and Capillary analyzers ABI-377 MegaBACE 1000

Topics for Today cont. Laboratory Information Management Systems (LIMS) General History Uses in a High Throughput Sequencing Facility Limitations Examples

Topics for Today cont. UPR Sequencing and Genotyping Facility Vision Status

The Cycle Sequencing Reaction REACTION OVERVIEW The Sanger sequencing method (a.k.a dideoxychain termination method) Copies a dna template by interrupting the replication process at one of the 4 bases (A,T,G,C) and producing enzyme labeled fragments of various sizes.

The Cycle Sequencing Reaction Components DNA template of unknown sequence Primer Taq Polymerase Deoxynucleotides (dNTP’s) Dideoxynucleotides (ddNTP’s) (modified)

The Cycle Sequencing Reaction Fragment Labeling Strategies Dye or radiolabeled ddNTP (Terminator) Dye or radiolabled dNTP (Internal labeling) Dye or radiolabled primer

The Cycle Sequencing Reaction REACTION OVERVIEW Fragment Labeling Strategies Dye or radiolabeled ddNTP (Terminator) MOST COMMON Dye or radiolabled dNTP (Internal labeling) Dye or radiolabled primer

The Cycle Sequencing Reaction REACTION OVERVIEW 4 process to remember in the reaction: Denature Anneal Extend Terminate

The Cycle Sequencing Reaction Interferences Too much Template can exhaust the supply of ddNTP’s Inappropriate primer dimers or hairpins can interfere with annealing GC rich templates (problems with denaturing) AT rich templates (small peaks)

The Cycle Sequencing Reaction Interferences Homopolymer regions (long strings of A’s,T’s,G’s,C’s) Templates with long repeats (microsatellites)

Sequencing Analyzers Slab Gel and Capillary Analyzers ABI-377 (Slab Gel) MegaBACE (Capillary)

ABI 377

+’s Easy to use Economic for Smaller Labs Instrument is not very expensive -’s Labor intensive Low volume of samples processed Takes up bench space (gels) Long run time (about 3.5 hours)

MegaBACE 1000 Capillary Sequencer Collect data from 96 sample at same time. Shorter Run Time (~2.0 hours) Needs Less Template Highly Sensitive

MegaBACE 1000

Sequence Analysis Tracks gel Files (ABI-377) Makes corrections for spacing, intensity, and overlap Determines sequence starting points Calls Bases Analyzed data is formatted for use in downstream analysis and sequence assembly software.

Data Collection and Storage LIMS (Laboratory Information Management Systems) Manages laboratory workflow Tracks and Stores Data from multiple instruments

Data Collection and Storage LIMS (Laboratory Information Management Systems) May have analysis features (sequence alignment, contig assembly, primer and probe design) Assists in local and public sequence database searches (GenBank, EMBL)

Data Collection and Storage Laboratory Information Management Systems (LIMS) History Molecular Biologist + data = Software Developer Developed, in-house, by organizations that needed help with their data. (hard work, long hours) Custom Built LIMS (private companies)

Data Collection and Storage Laboratory Information Management Systems (LIMS) History cont Turn-Key Systems (inflexible) Open System (flexible and functional)

Data Collection and Storage Laboratory Information Management Systems (LIMS) Uses in a High Throughput Sequencing Facility Staff and customers can easily track samples Allows quick distribution and access to results Quality Control Archives data for emergency retrieval Provides Security Business management tools (Billing)

Data Collection and Storage Laboratory Information Management Systems (LIMS) Limitations Keeping up with the technology of producing and analyzing many types of data Multiple formats (incorporating data from other sources) Cost (purchasing and modifying software, Fast and powerful hardware)

Data Collection and Storage Laboratory Information Management Systems (LIMS) Examples Life Science LIMS (Applied Biosystems) Manages lab process, generates reports, QC, Storage Gene Inspector (Textco) Sequence Analysis, searches data bases, lab notebook Geospia’s Finch Manages lab processes, generates reports, QC, analyzes data, Storage, searches public and private data bases.

Data Collection and Storage Laboratory Information Management Systems (LIMS) BOTTOM LINE No Perfect System

UPR - Sequencing and Genotyping Facility University of Puerto Rico Sequencing and Genotyping Facility

UPR - Sequencing and Genotyping Facility April of 2003 Objective: To provide the P.R. research community with automated DNA sequencing services

UPR - Sequencing and Genotyping Facility Other Services: Full Service Sequencing Genotyping (AFLP’S, Microsatellites, and SNP’s Troubleshooting and Analysis Assistance Emergency Retrieval of Data

UPR - Sequencing and Genotyping Facility Other Services: Primer selection Access to bioinformatics software

UPR - Sequencing and Genotyping Facility Approach: Phase 1: Creating a self-contained core resource. Phase 2: Promoting the Facility via a Web- based interface, training workshops, and targeted individual attention. Phase 3: Integrating the Facility with other established or planned core researchers facilities within the Biology Department.

UPR - Sequencing and Genotyping Facility Where we are now: Main Equipment (ABI-377, MegaBACE, Li Cor) Space Knowledge In Progress: Applying for funding Searching for Staff Developing SOP/ QC program Promotion

Collecting and Storing Sequences In the laboratory THANK YOU!!!