collecting and storing sequences in the laboratory heather helm upr sequencing facilities manager
TRANSCRIPT
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!!!