DNA Sequencing

 

The DNA Sequencing Group at the Partners HealthCare Center for Personalized Genetic Medicine (PCPGM) has a strong history of producing high quality, dependable, and informative results for collaborators and clients. Over the past 10 years, our focus has been to provide Sanger Sequencing support for our clients, including participating in the Human Genome Project by 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.
More recently, our focus has shifted to larger-scale Next-Generation sequencing using the Illumina HiSeq 2000, and the Illumina MiSeq Personal Sequencing platform. We have been involved in a wide variety of projects, including:

  1. Draft sequencing of unique strains of bacterial genomes (e.g., Pseudomonas aeruginosa and Vibrio cholera),
  2. Detecting mutations in bacterial genomes (e.g., Mycobacterium tuberculosis)
  3. Developing shRNA target screens from a variety of vendors,
  4. Constructing libraries and sequencing for whole transcriptome RNA-Seq of eukaryotic samples
  5. Detecting sequence for Reduced Representation Bisulfite Sequencing (RRBS)
  6. Constructing libraries of targeted regions using various selection methods.

We are actively developing library construction for Digital Gene Expression profiling and library construction for miRNAs.
All projects are managed through GIGPAD, our LIMS, and are taken on a first-come, first-served basis according to sample type and library construction method. Quality control is carried out for all processes and includes grouping of a PhiX sample with customer samples prior to library construction. All RNA-Seq and DGE library construction methods include incorporation of Ambion ERCC spike-in control mixes prior to cDNA synthesis to measure dynamic range and lower-limit of detection. Automation is used whenever possible, with the use of Pre- and Post PCR Biomek FX liquid handlers, Perkin Elmer Janus Automated Workstation, and Beckman Coulter SPRIworks library construction system.
For the HiSeq 2000, we guarantee 100 million clusters per lane of raw, unfiltered data. Any highly diverse library not reaching this level is offered a free rerun. Our turnaround time varies with the number of samples submitted in one batch; the fastest turnaround time is for groups of libraries filling a flow cell (7 lanes per flow cell).
For the MiSeq, we guarantee 10 million clusters per lane of raw, unfiltered data for the regular flow cell, the nano and micro flow cells will generate around 1 and 4 million clusters.
A standard analysis package is available for all libraries sequenced in our facility. This standard package includes the return of demultiplexed, unaligned, filtered reads in FASTQ format, as well as a summary statistic report. For RNA-Seq and DGE analysis, we offer a standard data package included in the per-lane sequencing charge that includes alignment of reads to a reference genome with TopHat, transcript abundance estimation with Cufflinks and differential expression analysis with Cuffdiff and CummeRbund. The report also provides several QC metrics for each sample. Additional and/or customized analysis should be discussed with us in advance, and a quote will be provided on a per-project basis. For shRNA methods we provide the researcher with demultiplexed counts of shRNA targets which appear in the library.
We welcome the opportunity to discuss projects with potential clients to ensure the selection of an appropriate method for their scope of work. We also pay close attention to changes in researcher focus and technology and welcome discussions with clients and companies for new technology testing and development, collaborative protocol development, and Beta-testing of instrumentation and software.

 

 

NEWS: After gaining several year experience using the Illumina GaIIx and HiSeq2000, PCPGM has added to it services the Illumina MiSeq v2 Personal Sequencer. While the HiSeq 2000 is more suited to the sequencing of larger genomes and transciptomes, the MiSeq offers faster turnaround times with lower coverage, making it ideal for quality control of libraries, whole exome analysis, whole genome sequencing of smaller model organism genomes, and amplicon sequencing.

Coverage (from Illumina specifications, using PhiX libraries)
25PE 5.5 hrs machine time, 750-850 Mb, > 90% Q30 average
150PE 24 hrs machine time, 4.5-5.1 Gb, > 80% Q30 average
250PE 39 hrs machine time, 7.5-8.5 Gb, > 75% Q30 average, analysis 2 hrs
Clusters 12-15 M (22-25 M future)

Regular chips ~15 M clusters
Nano Chips ~1 M clusters
Micro Chips ~4 M clusters
(Not guaranteed)

Please contact Alison Brown (abrown13@partners.org) or Dr. Sami Amr (samr@partners.org) for more information.