PCPGM DNA Sequencing Facility
Director: Amr, Sami, Ph.D.
Location: Partners HealthCare Center for Personalized Genetic Medicine, 65 Landsdowne Street, Room 350, Cambridge, MA 02139
The DNA Sequencing Group at the Partners HealthCare Center for Personalized Genetic Medicine has a strong history of producing high quality, dependable, and informative results for collaborators and clients. The DNA Sequencing Group participated in the Human Genome Project, building the STS-Based BAC map for Human Chromosome 12, and providing Chromosome 12 tiling path clones to the Baylor Human Genome Sequencing Center for sequencing.
The group sequenced 113 BACs for the Mouse Genome Project, contributing 24 megabases of finished mouse sequences to the published Mouse Genome, as well as providing draft sequences for unique strains of several bacterial genomes, including Pseudomonas aeruginosa, and Vibrio cholerae. More recently, the group participated in identifying mutations linked to numerous diseases, either in collaborations or by providing client laboratories with full service resequencing and analysis.
Director: Amr, Sami, Ph.D.
Phone: (617) 768-8377
Role: Lab Manager, PCPGM Genotyping Facility
Phone: (617) 768-8470
Phone: (617) 768-8557
|Facilities and Equipment:|
Location of Core: Partners HealthCare Center for Personalized Genetic Medicine, 65 Landsdowne Street, Room 350, Cambridge, MA 02139
- ABI 3730xl DNA analyzer
- Illumina Genome Analyzer
- Illumina MiSeq
- ABI capillary sequencing service
"This facility is equipped with ABI 3730xl DNA Analyzers. They provide reads of up to 800 high quality bases and produce approximately 1500 reads per day, per machine (1.2 Megabases of sequence per machine). The Biomek FX and Platemate robots are used to automate most steps for capillary sequencing.
- Automated primer design
- PCR amplification
- Sequencing reactions
- Sequencing detection, ABI 3730xl
- Data assembly
- Sequence variation identification and mutation detection using Phred,Phrap and Consed
- Fragment analysis (Please see Submission Requirements on the Fragment Analysis Service page)
- Chip-Seq service
Illumina Next Generation Sequencing can be used for the study of interactions between proteins and DNA via chromatin immunoprecipitation (ChIP-seq)
- Clinical diagnosis services
- Diagnostic test development
- Sequence analysis
- Sequencing for clinical diagnostics group
- Digital Gene Expression Service
Illumina Next Generation Sequencing can by used for expression profiling assays in bacteria or mammals using digital gene expression (GEX).
- Genomic sequencing service
- High throughput genome sequencing
"High throughput resequencing is supported for particular regions of interest in any genome, though most of the research core's work involves working with human, mouse, or bacterial genomes."
- Illumina next generation sequencing service
PCPGM offers next generation sequencing services on Illumina's HiSeq2000 and coming soon on the Illumina MiSeq. Both instruments use massively parallel sequencing-by-synthesis technology to generate DNA sequence data with unprecedented throughput and accuracy. More information can be found at Illumina Website.
- Mutation identification via resequencing service
"This facility provides full-service resequencing of regions of interest in one or more genomic DNAs, including the following:
- Discussion of the scope of the project and a cost quote
- Identification of genes in the region of interest as needed, with the Investigator
- Primer design using our automated system, to amplify desired regions
- Primer ordering
- QC of the primers on DNA standards, if required
- PCR amplification of DNA provided by Investigator
- PCR clean-up
- Sequencing reactions
- Sequencing reaction clean-up
- Sequence application to the ABI 3730 XL Analyzer
- Chromatograms are made available to Investigator over web (GIGPAD)
- Data assembly and analysis using Phred Phrap and PolyPhred
- One round of repeats and redesign if necessary
- Report of variations found throughout sequence
- Trouble shooting for 100% coverage if desired"
- Next gen seq bioinformatics support service
PCPGM, in addition to performing sequencing, performs standard bioinformatics analysis on all the samples it receives. The various options that are available to the customers, along with the requirements are listed below. Fulfilling all the requirements ensures timely return of processed data. Under each option the types of data that are deliverable and the mode of delivery are listed. The user must satisfy the requirements and mention the type of data, along with the mode of delivery with their order.
* RTA Intensity Data: The raw intensity files will be delivered in addition to the base calls. These base calls are in qseq format.
* Base Calls: Fastq formatted sequence files are generated for all base calls. This format can be easily converted to other formats.
* Base Calls+Alignment: ELAND is used to perform alignment on your samples. This will allow users to get an initial estimate on the quality of the data.
However to do this, the following information is required:
1. Reference Sequence
2. Any bias/vector sequence that we can expect in reads
3. If your reads are indexed, if so, how many bases, and location of the index (before or after the variable read)
4. If a separate primer was used for sequencing the index.
5. These set of information would help us properly extract any indexes, and trim vector sequences before aligning the read to the reference."
- Nucleic acid library construction service
"PCPGM offers library construction from:
* PCR products
* Genomic DNA
* ChIP-Seq products
* Total RNA
Sample Submission Requirements:
DNA/RNA Requirements for Library Construction:
Between 0.2 to 1 ug starting material is required, at a concentration of over 50 ng/ul as assessed by picogreen. The purity of the sample should be measured by the 260/280 OD ratio, and should be more than 1.8. A gel picture is required to measure the quality of the submitted sample.
Customer-Constructed Library Submissions:
The most common libraries sequenced at the PCPGM are SAGE/DGE, ChIP-Seq, pooled shRNA, concatenated PCR amplicons and Genomic shotgun libraries. Researchers are encouraged to QC and normalize their libraries to a concentration of between 20-100 nM, with a minimum volume of 20 ul, resuspended with clear buffers or water. Concentration should be assessed by picogreen quantitation only. A gel picture must also be provided to show the size of the library. All libraries must be purified using QIAquick PCR purification kit (Qiagen, part number 28104). No other kit can be substituted.
Before any sequencing or library construction can be carried out, a completed Sample submission form must be received. The form can only be used with a PC; Mac users should contact Alison Brown before attempting to complete the form. The appropriate forms should be completed and emailed directly to Alison Brown. Incomplete forms cannot be emailed and all required areas will be highlighted in red."
- PCR to detection service
For PCR to detection projects: Please contact us in advance to discuss your project needs. We may be able to serve larger projects but small projects will be referred elsewhere.
- Plasmid sequencing service
We can provide library creation services or you may submit your own libraries. Libraries are sequenced on the Illumina HiSeq2000. At least 10 million reads are generated per one lane of sequencing. If your library does not have an overrepresentation of few particular shRNA clones, up to 20 million reads could be generated per one lane of sequencing.
- Sequencing technology development service
- New technology testing and development
- Collaborative Protocol development
- Beta-test site for instrumentation and software
- shRNA library sequencing service
PCPGM offers resequencing services for shRNA pools with fast turnaround time using HiSeq2000 next generation sequencing platform. We employ a specific method developed in our laboratory for resequencing of shRNA pools. This methodology has significant advantages over the existing screening methods.
- Transcriptome and RNA-Seq service
PCPGM offers next generation sequencing services on Illumina's HiSeq2000 for total RNA at the transcriptome and digital gene expression level. Small RNA analysis will also be available - please contact us.
|Departmental Web Link(s):|