The DNA Sequencing Facility provides reliable, long read, automated Sanger sequencing with fast turnaround; microsatellite-based genotyping and fragment analysis; plasmid and BAC DNA preparation and purification; and related molecular biological services including PCR, cloning, sub-cloning, site-directed mutagenesis, and preparation of targeting vectors for gene targeting in mice. It also provides services and support for analysis and interpretation of sequence data as well as the design of approaches to complex sequencing projects.
For the last four years the facility has been providing Roche 454 sequencing service that includes library preparation, emulsion PCR and pyrosequencing for both genomic DNA and amplicons. Data analysis is provided in each project depending on the investigator’s specific need.
Ion Torrent's Personal genome machine (PGM) is the latest addition at the facility. Known for scalability, simplicity and speed, this inexpensive technology is advancing fast to achieve new goals in terms of throughput and read length. The maximum read length and the throughput available at this point is 200 b and 1 Gb respectively. The applications are similar to those of long-read 454 sequencer and includes targeted resequencing of barcoded samples, sequencing of captured library, sequencing of bacterial and viral genomes, sequencing of metagenomic samples, RNA-seq specially small RNA sequencing and validation of sequence data obtained on other platforms. The sequencer comes with Torrent Suite, the Torrent server analysis pipeline that is the primary software used to process raw data acquired by PGM sequencer to produce sequence read files. The base calls are in both SFF and FASTQ file formats for easy downstream analysis with third party analysis tools. The Torrent suite performs filtering, trimming, mapping with the generation of a Variant Caller report. This long read sequencer is going to bring down the cost of new generation sequencing significantly.
The range of services mentioned above along with the expertise of the facility personnel enables this core to provide full support for investigators at Penn, who can easily obtain fast, reliable data on genes of interest, whether they are doing targeted or whole genome tumor genome sequencing, deep resequencing, screening clones for sequences of interest, establishing the identity of new clones, or searching for mutations in specific genes.
"The 3100 Genetic Analyzer, a multi-color fluorescence-based DNA analysis system with 16 capillaries operating in parallel, offers high-quality data and efficient sample processing for the busy research lab."
The DNA sequencing facility is equipped with three ABI 3100 sequencers.
"The 96-capillary 3730xl DNA Analyzer is the Gold Standard for high throughput genetic analysis. Use this for DNA fragment analysis applications such as microsatellites, AFLP, SNP analysis, mutation detection and traditional DNA sequencing. Get the highest quality data at a low cost per sample."
The DNA Sequencing Facility is equipped with eight ABI thermal cyclers.
"The Agilent 2100 Bioanalyzer is a microfluidics-based platform for sizing, quantification and quality control of DNA, RNA, proteins and cells. Results are delivered within 30-40 minutes in automated, high quality digital data.
Miniaturization of analytical instrumentation has many advantages over conventional techniques. These advantages include improved data precision and reproducibility, short analysis times, minimal sample consumption, improved automation and integration of complex workflows. Launched in 1999, the Agilent 2100 Bioanalyzer was the first commercially available instrument to use microfluidics technology for the analysis of biological samples. Today, the Bioanalyzer offers a broad range of pre-validated analysis kits combined with an easy-to-use benchtop system."
Used for automated DNA extraction.
"Z™ Series COULTER COUNTER® Cell and Particle Counter uses the Coulter Principle (Electrical Sensing Zone Method) for sizing and counting particles"
"By incorporating a wide range of next-generation features into a small footprint design, Biomek NXP sets the standard for flexible laboratory solutions. It puts every aspect of liquid handling – including pipetting, dilution, dispensing and integration – into a single, automated system that is as powerful as it is efficient and economical. All while offering unprecedented options for changing needs."
"The Ion OneTouch™ System comprises two modules: the Ion OneTouch™ Instrument and the Ion OneTouch™ ES (enrichment system).
At the heart of the Ion OneTouch™ Instrument are three breakthrough technologies that enable automated delivery of templated Ion Sphere™ particles. The first is the reaction filter that creates millions of microreactors in which clonal amplification occurs. The second is the revolutionary inline PCR amplification plate that enables thermal cycling of the microreactors. The third is the integrated centrifuge, which recovers the templated Ion Sphere™ particles. Combined, these first-in-class technologies deliver massively parallel clonal amplification and recovery with walk-away operation—all in a small benchtop footprint.
The Ion OneTouch™ ES employs proven magnetic bead technology to isolate template-positive Ion Sphere™ particles that can be loaded directly onto the Ion semiconductor chip—delivering automated, highly reproducible enrichment with every run."
"Ion Torrent combines pre-existing semiconductor technology with a simple sequencing chemistry. The sequencer is essentially a solid state pH meter. Known for scalability, simplicity and speed, this inexpensive technology is really moving fast to achieve new goals in terms of throughput and read length.
The main applications of this technology at this point include:
• Resequencing of barcoded multiplexed samples,
• Sequencing of captured library (captured region 100 Kb to 1 Mb) using 316 chip
• Sequencing of bacterial and viral genomes
• Sequencing of metagenomic samples with 200 b sequencing chemistry
• RNA-seq specially small RNA sequencing
• Validation of sequence data obtained on other platforms
• Low cost sequencing of BAC clones and other constructs e.g. gene targeting vector"
"Ion Torrent Proton, an upgrade of PGM, combines pre-existing semiconductor technology with a simple sequencing chemistry (http://www.iontorrent.com). The sequencer is essentially a solid state pH meter. Known for scalability, simplicity and speed, this inexpensive technology is really moving fast to achieve new goals in terms of throughput and read length."
"The Pippin Prep facilitates library construction for the most popular NGS platforms, and is recommended by Illumina and Ion Torrent for certain workflows. The platform features the ability to collect narrow and even fragment distributions, as well as the flexibility to collect wide ranges of fragments with minimal effort. Target sizes or ranges of sizes are entered in software, and fractions are collected in buffer. Up to 5 samples per gel cassette may be run, with no possibility of cross contamination."
"The innovative QIAcube uses advanced technology to process QIAGEN spin columns, enabling seamless integration of automated, low-throughput sample prep into your laboratory workflow. No change of purification chemistry is required, assuring fast startup and immediate results. All steps in the purification procedure are fully automated – and up to 12 samples can be processed per run."
"QIAGEN TissueLyser (Tissue Lyser) provides rapid and efficient disruption of cells and tissues for the BioRobot. Processing of up to 2 x 96 samples takes as little as 2 – 4 minutes. Disruption is achieved through the beating and grinding effect of beads on the sample material as they are shaken with the sample in a variety of grinding vessels. The optimized design of TissueLyser Adapter Sets ensures that tubes remain securely sealed during disruption. This prevents cross-contamination, which is especially important for highly sensitive downstream applications, such as real-time RT-PCR or microarray analysis."
Enables highly sensitive fluorescence-based quantitation assays using dyes selective for dsDNA, RNA, and protein.
For 454 data storage and analysis
Cloning strategy is designed jointly by the user and the facility. Directional or non-directional cloning into any vector is performed by including right restriction sites in the primers, if necessary, for amplifying the insert for PCR-based cloning. After cloning the insertions sites are checked and orientations are determined by either restriction digestion and/or sequencing.
PCR products are cloned using Invitrogen's TA cloning system or any other vector as requested.
After designing the cloning strategy, the insert is retrieved from the donor vector and appropriate restriction sites are created if necessary. The insert is then cloned into appropriate recipient vector followed by the screening for correct clones by restriction digestion and/or sequencing.
We also offer ligation-independent cloning (LIC) when requested by the user."
"Gene targeting, a methodology for the generation of knock-out and knock-in mice involves construction of a targeting vector (done by this facility), electroporation of embryonic stem (ES) cells with the construct and selection of homologous recombinant ES clones with southern blotting (done by Gene Targeting Service), injection of recombinant cells into blastocysts and generation of chimera (done by Transgenic Mouse Facility) and finally germ line transmission to develop targeted mouse cell lines.
DNA Sequencing Facility constructs targeting vectors for both conditional knock-out and knock-in mutations using BAC recombineering method of Dr. Neal Copeland.
Ref: Liu et. al. 2003, 13:476-484, Genome Research
• The vector design is to be provided by the investigator. Gene Targeting Service designs vectors for a fee.
The DNA Sequencing Facility will
• order 2 BAC clones if available
• design and order primers for homology arms (300 – 600 bases) of the retrieval vector and the mini targeting vector(s)
• make retrieval vector and retrieve genomic target from the BAC DNA by homologous recombination
• make 1 or 2 mini targeting vectors as required
• in the case of knock-in construct introduce mutation(s) in one of the homology arms
• introduce loxP or FRT sites into the target using Cre or Flpe recombinases
• do maxi DNA prep of the final vector
• verify sequence of the final vector including all exons in the target, two ends of neo gene and LoxP and FRT sites
We also make probes for southern blotting & purify construct DNA for direct microinjection into mouse oocytes."
"The DNA Sequencing Facility currently offers the DNA 1000, High Sensitivity DNA and RNA 6000 Pico assays with rapid turnaround time. If you are interested in an assay we do not offer, please contact the facility directly."
"Genotyping/Fragment sizing is currently done on ABI (Applied Biosystems) 3130XL capillary sequencer, for microsatellite-based genotyping used in linkage analysis, identifying VNTR alleles in a genetic screen, mapping genomic clones, or any other purposes. For this PCR products need to be generated with one primer labeled on the 5′ side preferably with an ABI synthesized dye FAM, HEX (or VIC), NED etc (see below). Submit about 3-4 μl PCR products (single or pooled) in 0.2 μl strip tubes or in a reaction plate. Samples can also be submitted in a ready-to-load format (PCR products + ROX size standard + formamide already added) on an ABI 96-well reaction plate."
High Throughput Sequencing of PCR products or plasmid DNA
Users can submit a full 96-well PCR plate (compatible with the ABI PCR machine) with prescribed amount of DNA (purified PCR products or plasmid DNA) and primer in each well and send the sample information following a special format in excel by e-mail.
High Throughput Sequencing from Bacterial Cultures (high copy plasmids only)
The facility will prepare plasmid DNA from cultures submitted in a deep-well plate and sequence both ends. A culture plate can also be prepared from a glycerol stock plate. The sample information is to be sent following a special format in excel by e-mail. This method works for high copy plasmids only."
"Torrent Suite, the Torrent server analysis pipeline is the primary software used to process raw data acquired by PGM/Proton sequencer to produce read files containing high quality bases. The base calls are in both SFF and FASTQ file formats for easy downstream analysis with third party analysis tools. The Torrent browser provides many matrices, graphs and reporting features derived from the pipeline results.
Filtering and trimming: This is done to remove low quality bases or uncertain base calls by filtering out entire reads and trimming low quality 3’ ends of reads respectively. The SFF files contain per-base quality scores along with all other read information. The FASTQ files also provide per-base quality scores.
TMAP: Torrent Mapping Alignment Program implements a two-stage mapping approach – reads that do not align during the 1st stage are passed to the 2nd stage with a new set of algorithms and/or parameters. Overall the alignment provides an index to determine run and library quality.
Variant Caller: After analysis the variant caller generates a report of the SNVs and the insertion-deletions obtained from the data set (Torrent Suite User Documentation, Life Technologies).
Commercial software NextGene, DNASTAR, PARTEK and others are available for analysis of PGM/Proton data.
We provide data analysis service for a fee using Torrent suite, NextGene and other software."
Several off-the shelf targeted gene panels available:
• AmpliSeq Cancer Hotspot Panel v2
• Comprehensive Cancer Panel
• AmpliSeq Inherited Disease Panel
• AmpliSeq Custom Panel "Ion Torrent combines pre-existing semiconductor technology with a simple sequencing chemistry ( http://www.iontorrent.com ). The sequencer is essentially a solid state pH meter. Known for scalability, simplicity and speed, this inexpensive technology provides an array of options from 500K reads on a 314 chip to 5M reads on a 318 chip as well as a read length of 200 and 400b. The sequencing runs are only 4 – 8 hours compared to few days run on Illumina. Only one sample is run at a time. So there is no waiting necessary to fill out a plate, a flow cell or a slide unlike other new generation sequencing platforms."
"Ion Torrent Proton, an upgrade of PGM, combines pre-existing semiconductor technology with a simple sequencing chemistry (http://www.iontorrent.com). The sequencer is essentially a solid state pH meter. Known for scalability, simplicity and speed, this inexpensive technology is really moving fast to achieve new goals in terms of throughput and read length.
Applications on PI chip include: AmpliSeq Transcriptome and Total RNA-Seq; AmpliSeq Exome and TargetSeq Exome; Large Targeted Gene Panel e.g. Comprehensive Cancer Panel, Inherited Disease Panel and Custom Panel.
Only 1 sample is run at a time. No waiting is necessary for a plate/flow cell/slide to fill up."
"The facility will sequence through DNA clones by primer walking or, if needed, deletion subcloning. Please fill out a Request for Sequencing Project Management form when submitting the sample, indicating if you wish to select primer sequences yourself or delegate the responsibility to facility staff. Charges for primer walks are calculated according to the number of lanes sequenced and primers constructed. A clean-up treatment of sufficient template for the whole project (nominally 4-5 µg per kb for each direction) is recommended to produce quality sequence and maximize step size.
After good quality template is obtained, each step (~500 nucl.) requires around one week, including primer selection and synthesis, sequencing, sequence evaluation, and assembly. Charges for deletion subcloning should be discussed with facility staff. Assembly of the overall sequence, ORF analysis, and homology searches can be performed, at no charge, if requested by the user."
"PCR, PCR clean up & Cloning
PCR of genomic DNA or BAC DNA is done using custom primers. Primers can also be designed at the facility. The optimization and the clean up of PCR products are done using various procedures. The cleaned up products can be directly processed for Sanger sequencing at the facility or used for cloning.
Transformation and Cloning
Design of cloning strategy followed by directional or non-directional clonings are performed using appropriate competent cells and vectors. Insertions sites are checked and orientations are determined by either restriction digestion and/or sequencing.
PCR products are cloned using Invitrogen’s TA cloning system.
After designing the cloning strategy, DNA is sub-cloned into an appropriate expression vector followed by the preparation of the construct DNA for micro-injection into mouse zygotes.
Mutagenesis including point mutation, insertion and deletion of single or multiple amino acids and large insertion are done using Stratagene’s Quick Change kit. This includes design and synthesis of the mutagenic primers, mutagenesis and verification by sequencing. The synthesis and the PAGE purification of the primers are outsourced to Invitrogen.
AMPure Bead Purification of Amplicons and DNA Fragments
Agencourt (Beckman) AMPure XP bead-based DNA purification of amplicons efficiently removes unincorporated dNTPs, primers, primer dimers, salts and other contaminants, resulting in high quality DNA suitable for sensitive downstream applications such as sequencing, genotyping, PCR, and sub-cloning. Recovery: 70 - 90% of double stranded and single stranded DNA with fragment sizes 150 bp - 10 kbp. This is the recommended purification method during sample preparation in most protocols for various new generation sequencing platforms."
"Rapid genotyping of transgenic mice is highly important for successful breeding as well as for reducing the cost of strain maintenance. Particularly for large and multiple litters one needs to have the genotyping performed quickly in a cost-effective manner.
We offer a fast and convenient service for large scale mouse genotyping using Sigma REDExtract-N-Amp Tissue PCR kit starting from tail biopsies or ear punches. The user provides the mouse tails, control DNA with the transgene in it, and the PCR primers for the transgene together with any special PCR protocol. Two PCRs are done for each sample, one for the transgene and the other a control mGAPDH fragment. The amplicons are run on agarose gel and the labeled gel electrophoresis picture is provided within 48 hrs."
"PCR of genomic DNA or BAC DNA are done using custom primers. Primers can be designed at the facility. The optimization and the clean up of PCR products are done using various procedures including exo-sap, column and gel purification. The cleaned up products can be directly processed for Sanger sequencing at the facility or used for cloning.
PCR products are cloned using Invitrogen's TA cloning system."
"The facility uses two ABI (Applied Biosystems) sequencers: one 96-capillary 3730XL and one 16-capillary 3130XL sequencer. The sequencing is done on 3730XL with BigDye Taq FS Terminator V 3.1. The genotyping and fragment sizing are done on 3130XL;. Please consult our recommendations for template preparation to avoid failures. Standard primers (listed below) are provided by the facility at no charge. Custom primers are to be provided by the user, the facility no longer synthesizes primers.
Compared to the previous generation ABI 3700 sequencer, the 3730XL offers enhanced data quality and more successful samples per run, minimum reagent and sample consumption, high reliability, easy maintenance, and automated operation with accurate sample tracking. It generates enormous amounts of information faster and at a lower cost. The sequence length of read (LOR) is routinely more than 700 bases with < 2% error or ambiguity. Users can either choose to have the full sequencing reaction done at the facility, or provide ready-to-load samples (reaction and purification done by user). Our latest offer is high throughput DNA Sequencing at a low introductory price. Please use the relevant link for more information.
The turn around time has recently been reduced to one working day for all samples submitted by 3:30 PM with only occasional exceptions due to staff shortage, very high demand, and/or equipment failure. More and more emphasis is being put on the quality of the sequencing data. Every sequence result is looked at for overall quality, and accuracy of the base call. Whenever necessary, the samples are either reanalyzed with a different base caller and the bases edited, or reloaded to improve the quality. In some cases, samples are resequenced at no extra cost. Network licenses for the most common sequence analysis software like Mac Vector, Sequencher, Vector NTI are available from the Penn Molecular Profiling Group. Facility staff will consult with users whenever there are problems, and will make suggestions to improve sequencing results for regular and difficult templates.
As indicated on the sequencing submission site and the Submission Guide, please provide only requisite amount of template (or template + primer mix) for each reaction in a separate conjoined 0.2 ml strip tubes. This is necessary so that the samples can be processed on Biomek robot. Users may request that templates and primers be kept at the facility if they are being used in an active sequencing project."
"Sequence Assembly & Analysis
Assembly of sequences into a contig, identification of ORFs, analysis of properties of the predicted protein, and other analyses can be performed at the facility using MacVector and other software.
These services are included at no charge for large sequencing projects, and they are available at no charge to Cancer Center and Department of Genetics members.
BLAST, BLOCKS, BLITZ, PROSITE and FASTA searches are performed using WWW or e-mail servers at no charge. Please contact the facility to arrange for database searches or for training.
Genomic sequences can also be processed to identify interspersed repeats, low complexity regions, GRAIL exons, transcription factor binding sites, frequency of mono, di, and tri nucleotides, and other features. Genomic sequences can also be submitted to the GAIA genome annotation system at the Bioinformatics Laboratory.
Facility staff will also submit sequences to Genebank if requested by the investigator."
"Mutagenesis including point mutation, insertion and deletion of single or multiple amino acids and large insertions are done using Stratagene's QuickChange Lightning kit. This includes design and synthesis of the mutagenic primers, mutagenesis and verification by sequencing. After mutageneis the DNA can be maxi prepped if requested. Simultaneous mutagenesis at different sites (Multisite mutations) can also be done using Stratagene's multi site-directed kit."
"In NGS precise size selection helps ensure uniformity of read lengths and reduces the impact of short fragments on total sequencing throughput. The facility performs size selection of DNA fragments with a target size range between 90bp and 1.5kb using the Sage Science Pippin Prep instrument."
"Template DNA can be prepared and/or purified by the facility staff. Plasmid, cosmid, lambda phage, and BAC preps are offered through the Sequencing Facility. PCR reactions and purification using commercial columns is also offered by the facility. Plasmid minipreps are done on the Qiagen BioRobot. These preps yield 5-10 µg DNA for high copy number plasmids. If used for sequencing, the Facility guarantees the results, i.e. we will repurify and repeat sequencing at no additional charge until successful sequence is obtained.
The facility also offers reliable treatment of marginal or bad templates to make them suitable for automated sequencing. This involves RNase treatment followed by column purification or phenol-chloroform extraction. This treatment improves quality of template, producing more reliable sequence and longer reads. For primer walking projects we recommend that a substantial amount of DNA (nominally 4-5 µg per kb for each direction) be treated this way and be used for the whole project.
Templates prepared or purified by the facility staff are guaranteed to produce good sequence with standard, reliable primers, barring sequence specific problems (GC rich templates, secondary structure, internal repeats). The facility also provides free training in template preparation and troubleshooting, at users' request."
"GeneMapper® Software is a flexible genotyping software package that provides DNA sizing and quality allele calls for all Applied Biosystems electrophoresis-based genotyping systems. This software specializes in multiapplication functionality, including amplified fragment length polymorphism (AFLP®), loss of heterozygosity (LOH), microsatellite, and SNP genotyping analysis. GeneMapper® Software can help users increase data processing efficiency with remote auto-analysis and command line operation, and allows for multiuser, client-server deployment. The software uses Process Quality Values (PQVs) for automated identification that reduces data review time for high throughput genotyping. In addition, the security and audit features help users meet 21 CFR 11 requirements."
"Data from the Ion semiconductor sequencer streams automatically to the Torrent Server, which contains Torrent Suite Software. Torrent Suite Software processes the raw voltage semiconductor sequencing data into DNA base calls. This process happens without user intervention and can be complete in as little as 30 minutes following the conclusion of the sequencing run."
"MacVector is a comprehensive Macintosh application that provides sequence editing, primer design, internet database searching, protein analysis, sequence confirmation, multiple sequence alignment, phylogenetic reconstruction, coding region analysis, and a variety of other functions. MacVector is widely regarded as the most intuitive, easy to use program available for sequence analysis.
• graphical sequence editing
• gateway and topo cloning
• auto annotation
• primer design
• DNA analysis
• protein analysis
• database searching
• multiple sequence alignment
• sequence assembly"
"NextGENe is the perfect analytical partner for the analysis of desktop sequencing data produced by the ION PGM™, Roche Junior, Illumina MiSeq as well as high throughput systems as the Roche FLX, Applied BioSystems SOLiD™ and Illumina® GA, and HiSeq systems. NextGENe software, which runs on a Windows® Operating System, provides a biologist-friendly point & click interface that does not require scripting or other bioinformatics support, often required when using programs such as CLC Bio, LaserGene, NGEN, MAQ,SOAP,Top Hat, BWA and Bowtie.
NextGENe software employs unique sequencing platform specific technologies in one free-standing multi-application package:
• SNP/INDEL & Structural Variant Analysis
• Prediction and Rare Disease Discovery
• Whole Genome Alignment
• Transcriptome/RNA-SEQ: Expression and Alternative Splicing Analysis
• miRNA, ChipSeq, Serial Analysis of Gene Expression (SAGE) & Digital Gene Expression (DGE) Analysis
• de novo assembly
• Targeted Re-Sequencing
• Forensic Profiling
• Automation Pipeline"
"The GS Data Analysis Software package includes the tools to investigate complex genomic variation in samples including de novo assembly, reference guided alignment and variant calling, and low abundance variant identification and quantification. The suite of software is provided with the GS Junior and GS FLX System at no additional cost and allows researchers to begin interpreting sequence data immediately, without the need to invest in complex and expensive third party solution. Each of the software tools incorporates flow and signal information into the sequence analysis algorithms leading to higher confidence variant calling. Additionally, researchers can interrogate sequence data down to the flow-by-flow signal intensities used in base calling."
DNA sequencing analysis software.
"Vector NTI® software is a completely integrated suite of sequence analysis and design tools that help you manage, view, analyze, transform, share, and publicize diverse types of molecular biology data, all within one graphically rich analysis environment.
• Curate—store and manage collections, visualize maps, and search sequences
• Discover—analyze, compare, and contrast sequences
• Design—cloning strategy; primers for PCR, cloning, and resequencing; and gel simulations of sequences
• Confirm—contig assembly, sequence validation, and literature validation"