Collecting and Storing Sequences

In the laboratory

Heather HelmUPR Sequencing Facilities Manager

Topics for Today The Cycle Sequencing Reaction

Reaction Overview Interferences Sequencing Analyzers

Slab Gel and Capillary analyzersABI-377MegaBACE 1000

Topics for Today cont. Laboratory Information Management

Systems (LIMS)General HistoryUses in a High Throughput Sequencing FacilityLimitationsExamples

Topics for Today cont.

UPR Sequencing and Genotyping FacilityVisionStatus

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

The Cycle Sequencing ReactionREACTION OVERVIEW

4 process to remember in the reaction: Denature Anneal Extend Terminate

The Cycle Sequencing ReactionInterferences 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 ReactionInterferences

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

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

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 StorageLIMS (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 StorageLaboratory Information Management Systems

(LIMS)

HistoryMolecular 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 StorageLaboratory Information Management

Systems(LIMS)

History cont Turn-Key Systems (inflexible) Open System (flexible and functional)

Data Collection and StorageLaboratory 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 StorageLaboratory 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 StorageLaboratory 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 StorageLaboratory Information Management

Systems (LIMS)

BOTTOM LINE

No Perfect System

UPR - Sequencing and Genotyping Facility

University of Puerto RicoSequencing 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!!!