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NextGen Sequencing Core (MGH)

Director: Sadreyev, Ruslan, Ph.D.

Location: Simches Research Center, 185 Cambridge St., Boston, MA 02114

Summary:

The NextGen Core is a collaboration between the Department of Molecular Biology, the Center for Human Genetics Research, the Center for Computational Biology, and the Executive Committee on Research (ECOR).

Currently, the Core operates using a single Ilumina HiSeq instrument, accompanied by Illumina's cBot for cluster generation. This upgrade from our Genome Analyzer II doubled our capacity and greatly increased the data amount, quality, and stability over extra-long reads.

The Core is located in the state-of-the-art Richard Simches Research Center on Cambridge St. as part of the MGH main campus in Boston. The many multi-investigator groups in the building - including those that study human genetics, stem cells, genomics, and more - make it the perfect location for the Core to service the researchers in those groups. The majority of customers come from MGH, but we also service customers at other academic medical centers and industry.

Affiliations:

People:

Resources:

Instruments

  • Agilent Bioanalyzer ( Microfluidics platform )

    "The Agilent Bioanalyzer is a microfluidics-based platform for sizing, quantification and quality control of DNA, RNA, proteins and cells."

  • Covaris S2 ( Sample preparation system )

    "The Covaris S-series are advanced instruments based on technologies developed from both medical diagnostic imaging and therapeutic ultrasound industries. Covaris has developed numerous applications for the biological laboratory; using our S-series products, many processes can be completed within seconds. "

  • Illumina HiSeq 2000 ( DNA sequencer )

    "HiSeq 2000 makes it possible for individual labs to take on the largest and most complex sequencing studies at the lowest cost. The ability to process larger numbers of samples and to decode larger and more complex genomes means that virtually any sequencing project is now within reach."

Protocols

  • Agencount AMPure XP PCR Purification System ( Protocol )

    "The Agencourt AMPure XP PCR1 Purification systems utilize Agencourt’s solid-phase paramagnetic bead technology for high-throughput purification of PCR amplicons. Agencourt AMPure XP utilizes an optimized buffer to selectively bind PCR amplicons 100bp and larger to paramagnetic beads. Excess primers, nucleotides, salts, and enzymes can be removed using a simple washing procedure. The resulting purified PCR product is essentially free of contaminants."

  • Agilent SureSelect Target Enrichment System Protocol ( Protocol )

    "This guide describes Agilent's recommended operational procedures to capture genomic regions of interest using Agilent's SureSelect Target Enrichment System Kit and sample preparation kits for next- generation sequencing. This protocol is specifically developed and optimized to use Biotinylated RNA oligomer libraries, or Bait, to enrich targeted regions of the genome from repetitive sequences and sequences unrelated to the research focus. This guide uses the Illumina single- end sequencing platform for library preparation."

  • ChIP-Seq Sample Prep ( Protocol )

    "This protocol explains how to prepare libraries of chromatin-immunoprecipitated DNA for analysis on the Illumina Cluster Station and Genome Analyzer. You will add adapter sequences onto the ends of DNA fragments to generate the following template format:"

  • Covaris DNA Shearing Fragments <1.5Kb ( Protocol )

    "Covaris DNA Shearing Fragments <1.5Kb"

    "Methods are transferable between the S2 system and the automated E210 (batch) system."

  • DSN Normalization Protocol (for RNA-Seq libraries) ( Protocol )

    "This protocol explains how to normalize Illumina® RNA-seq sample
    preparation based on the use of the Duplex-Specific thermostable nuclease (DSN) enzyme, purified from Kamchatka crab hepatopancreas and manufactured by Evrogen (www.evrogen.com). DSN normalization is performed after RNA-seq sample preparation and before cluster generation. It involves the degradation of abundant DNA molecules derived from rRNA, tRNA, and housekeeping genes while preserving DNA molecules derived from less abundant transcripts. This method can be useful in a wide range of applications, including transcriptome discovery and annotation, the analysis of bacterial transcriptomes that lack poly-A tails, and the analysis of highly degraded RNA from sources such as FFPE."

  • Genomic DNA Sample Prep ( Protocol )

    "This protocol explains how to prepare libraries of genomic DNA for analysis on the Illumina Cluster Station and Genome Analyzer. You will add adapter sequences onto the ends of DNA fragments to generate the following template format:"

  • GEX DpnII Sample Prep ( Protocol )

    "This protocol explains how to prepare libraries of mRNA for subsequent cDNA tag sequencing on the Illumina Cluster Station and Genome Analyzer. You will isolate mRNA and create 20 bp tags with adapter sequences ligated onto the ends of the cDNA fragment to generate the following template format:"

  • GEX NlaIII Sample Prep ( Protocol )

    "This protocol explains how to prepare libraries of mRNA for subsequent cDNA tag sequencing on the Illumina Cluster Station and Genome Analyzer. You will isolate mRNA and create 21 bp tags with adapter sequences ligated onto the ends of the cDNA fragment to generate the following template format:"

  • MatePair v2 2-5kn Sample Prep ( Protocol )

    "This protocol explains how to prepare 2–5 kb mate pair genomic DNA libraries for cluster generation and analysis using the Illumina Mate Pair Library Preparation Kit v2. The v2 kit is formulated to generate 10 mate pair libraries with a gap size ranging from 2–5 kb and includes several modifications to the first version of the Illumina Mate Pair Library Preparation Kit. The modifications, which increase protocol robustness, improve usability, and generate higher quality and more diverse libraries, include:

    * Combined end repair and biotinylation reaction
    * Using QIAEX II suspension instead of QIAquick spin columns, because of its superior performance in isolating large DNA fragments
    * Improved size selection and gel running conditions
    * Increased circularization volume and amount of size-selected DNA used in circularization reaction
    * Recommendations for using the Covaris S2 shearing device
    * Changes to the streptavidin bead wash procedure"

  • mRNA-Seq Sample Prep ( Protocol )

    "This protocol explains how to convert total RNA into a library of template molecules suitable for high throughput DNA sequencing for subsequent cluster generation.

    The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. Then the cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNaseH. These cDNA fragments then go through an end repair process, the addition of a single ‘A’ base, and then ligation of the adapters. These products are then purified and enriched with PCR to create the final cDNA library."

  • Multiplexing Sample Prep ( Protocol )

    "This protocol explains how to prepare libraries of DNA fragments for multiplexed paired-end or single-read sequencing on the Illumina sequencing platform. You will add adapter sequences onto the ends of DNA fragments to generate the following template format:"

  • NextEra Epicenter Transposon-Based Library Preparation ( Protocol )

    "The Nextera™ DNA Sample Prep Kit is designed to prepare genomic DNA libraries compatible with the Illumina® Genome Analyzer I and II and Hi-Seq™ 2000 sequencers. Nextera technology employs in vitro transposition to simultaneously fragment and tag DNA in a single-tube reaction, and prepare sequencer-ready libraries in under 2 hours. The Nextera library preparation procedure is a significant improvement upon current procedures, which generally consist of distinct DNA fragmentation, end-polishing, and adaptor-ligation steps. The Nextera library preparation procedure combines these steps into one (tagmentation), uses only 50 ng of starting DNA, and allows incorporation of platform-specific tags and optional barcodes."

  • Paired-End Sample Prep ( Protocol )

    "This protocol explains how to prepare libraries of genomic DNA for paired-end sequencing analysis. The goal of this protocol is to add adapter sequences onto the ends of DNA fragments to generate the following sequencing library format:"

  • SmallRNA v1.5 Sample Prep ( Protocol )

    "This protocol explains how to prepare small RNA libraries using the alternative v1.5 for subsequent sequencing during cluster generation. Libraries prepared by this method should be loaded only on single-read flowcells for cluster generation.

    This protocol is designed to use either total RNA or purified small RNAs as input. You ligate the adapters necessary for use during cluster creation, reverse-transcribe, and PCR amplify to generate the following template:"

  • TruSeq DNA Sample Prep ( Protocol )

    "This protocol explains how to prepare 12 pooled indexed paired‐end libraries of genomic DNA (gDNA) for subsequent cluster generation and DNA sequencing using the reagents provided in the Illumina ® TruSeq™ DNA Sample Preparation Kit. The goal of this protocol is to add adapter sequences onto the ends of DNA fragments to generate multiplexed single read or paired end sequencing libraries."

  • TruSeq Exome Enrichment Guide ( Protocol )

    "This protocol explains how to capture exome sequences of a human DNA library that was prepared using the Illumina® TruSeq™ DNA Sample Prep Kit. Reagents provided in the TruSeq Exome Enrichment Kit subsequently prepare the library for sequencing targeted regions on the Illumina sequencing platform. The goal of this protocol is to enrich for exome sequences in solution using two rounds of hybridizations."

  • TruSeq RNA Sample Prep ( Protocol )

    "This protocol explains how to convert the mRNA in total RNA into a library of template molecules suitable for subsequent cluster generation using the reagents provided in the Illumina ® TruSeq™ RNA Sample Preparation Kit. The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then go through an end repair process, the addition of a single ‘A’ base, and then ligation of the adapters. The products are then purified and enriched with PCR to create the final cDNA library."

  • TruSeq Small RNA Sample Prep ( Protocol )

    "The Illumina® TruSeq™ Small RNA Sample Preparation protocol is used to prepare a variety of RNA species. The protocol takes advantage of the natural structure common to most known microRNA molecules. Most mature miRNAs have a 5ʹ-phosphate and a 3ʹ-hydroxyl group as a result of the cellular pathway used to create them. Because of this, the Illumina adapters in this kit are directly, and specifically, ligated to miRNAs.

    This guide explains how to prepare libraries for subsequent cluster generation, using total RNA or purified small RNA as input. The protocol describes the steps for adapter ligation, reverse transcription, PCR amplification, and pooled gel purification to generate a library product."

Services

  • Data Analysis ( Data analysis service )

    "Illumina's HiSeq software automatically makes base calls and generates FASTQ sequence and quality files, which are delivered to you. As a separate service, the core's experienced bioinformatics team can perform custom alignment and downstream analysis relevant to your project. For this service contact bioinfo@molbio.mgh.harvard.edu"

  • Library Validation ( Material analysis service )

    "We strongly recommend that all libraries are validated and quantitated using Bioanalyzer, qPCR, or similar methods before submission. In addition, the core staff validates all submitted libraries before sequencing to ensure maximum quality and cluster yield. This service is included in the sequencing cost. Please click the "library validation" box in Galaxy when submitting your samples if you are submitting barcoded subsamples and need us to pool them for you."

  • NextGen sequencing ( Material analysis service )

    "Up to 7 samples (plus a control) may be run on a single flow cell, or multiple samples may be multiplexed in one lane if obtaining the absolute highest coverage is not necessary or if previous runs have yielded excessive coverage (this may reduce costs). When deciding, keep in mind that our HiSeq is currently putting out approximately 150 million reads. Our core is actively engaged with Illumina scientists and other NextGen sequencing leaders to ensure we are up-to-date with reagents, software, and parts, and quality is constantly monitored throughout the run.

    We currently offer:

    • 50 cycle single read or paired end reads
    • 100 cycle single read or paired end reads
    • Multiplexing for any read type"


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Last updated: 2015-11-19T12:48:43.081-06:00

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