Next-Generation Sequencing Core (Penn)

Director: Kaestner, Klaus, Ph.D.

Summary:

The NGSC offers ultra high throughput sequencing services for the PSOM research community. We offer library quality assessments, sequencing, and optional preliminary data analysis for a wide variety of experimental protocols including ChIP-seq, RNA-Seq, HITS-CLIP, miR-Seq, exome capture, and BIS-seq. We offer limited library preparation services, but can advise on library preparation techniques. We have two Illumina hiSeq2000s for large-scale sequencing and a MiSeq for sample evaluation or library testing. To get started, visit our website, create an account for yourself, then create a new experiment and we will contact you.

Affiliations:

• Perelman School of Medicine

People:

• Director: Kaestner, Klaus, Ph.D.
  

Member: Schug, Jonathan, Ph.D.
Role: Technical Director, Next-Generation Sequencing Core

Resources:

Instruments

• Agilent BioAnalyzer ( Microfluidics platform )
  

  "The NGSC uses the Agilent bioAnalyzer to assess the quality and quantity of samples and libraries."

• Agilent Bravo ( Liquid handler )
  

  "Automated Liquid Handling Platform."

• Covaris LE220 ultra-sonicator ( Sonicator )
  

  "Focused-ultrasonicator for preparing genomic DNA, ChIP-Seq chromatin, extracting NA from FFPE samples."

• Fluidigm C1 ( Cell assay platform )
  

  "Automated solution for single-cell genomics research - WGA, RNA-Seq, & Taq-Man."

• Illumina HiSeq 2000 ( DNA sequencer )
  

• Illumina HiSeq 2500 ( DNA sequencer )
  

  "- 250 million 50, 75, 100, 125 single or paired reads
  - 150 million 250PE reads in rapid mode"

• Illumina miSeq ( DNA sequencer )
  

  "1, 4, or 20 million 50, 75, 150, or 300bp single or paired end reads."

• Illumina NextSeq 500 ( DNA sequencer )
  

  "- 400 million 75SR, 75PE, 150PE reads
  - 130 million 75PE or 150PE reads"

• Pippin Prep ( Gel electrophoresis system )
  

  "The Pippin Prep is a preparative electrophoresis platform that separates and extracts DNA fragments. Using pre-cast and disposable gel cassettes, DNA is automatically collected in buffer according to software-input size ranges. DNA fractions (typically adapter-ligate NGS libraries) are removed using standard pipettes. Workflow requires about 1-2 minutes of hands on time per sample, and cassettes are available for extractions between 50 bp and 8kb. Typical run times (four samples per run) are between 50 and 100 minutes."

• Qubit fluorometer ( Fluorometer )
  

  "The NGSC uses the Life Technologies QuBit to assess the concentration of libraries. The Qubit can measure protein, single- and double-stranded DNA, and RNA. The single-stranded DNA mode is especially helpful to quantify RNA-seq libraries with the 'high junk'.
  
  This high junk is good library and is an indication of over PCRing or not enough primers. Since the ends of the library have sequence homology, they will substitute for the primers and thus cause daisy chaining or a big clump of ssDNA. This is the high junk. Therefore, denaturing the libraries and using the ssDNA Qubit kit allows us to resolve the actual concentration of these complicated libraries."

Protocols

• ChIP chromatin preparation protocol ( Protocol )
  

• Chromatin preparation from small cell populations protocol ( Protocol )
  

• NGSC RNA-Seq protocol ( Protocol )
  

  Protocol used in Next-Generation Sequencing Core for RNA-Seq.

• RNA extraction protocol ( Protocol )
  

  Protocol describes RNA extraction procedure from cells, islets or small amounts of tissue (<30mg) and from animal tissues (150 to 1000 mg).
  
  Prepared by Peter White, Ph.D.

• RNA-Seq library preparation (Clontech) protocol ( Protocol )
  

  "The SMARTer Ultra Low RNA Kit allows high-quality cDNA synthesis starting from as little as 100 pg of total RNA or cells. The kit has been designed and validated to prepare cDNA samples for sequencing and quantitation with the Illumina HiSeq and Genome Analyzer sequencing instruments."

• RNA-Seq library preparation (NuGEN) protocol ( Protocol )
  

  "The Ovation® RNA-Seq System V2 provides a fast and simple method for preparing amplified cDNA from total RNA for RNA-Seq applications (transcriptome sequencing)."

• rRNA depletion for Illumina TruSeq protocol ( Protocol )
  

Services

• High-throughput sequencing service ( Material analysis service )
  

  Sequencing options include:
  
  • Single-end or paired-end sequencing
  • read length: 50, 75, 100, 125, 150, 250, 300 bp
  • depth: from 1 to 400 million reads or pairs

• Initial analysis of sequencing data ( Data analysis service )
  

  "We offer a complete set of initial analyses for most techniques. The initial analysis usually covers cleaning and trimming, alignment, and quantification. In many cases, differential analyses are available.
  
  Analyses available include:
  
  • ChIP-Seq: Peak calls, list of target genes
  • miR-Seq: Expression levels for known miRNAs
  • RNA-Seq: Expression levels for known genes, exon junctions, coverage
  • HITS-CLIP: Position and intensity of footprints on known transcripts and genome
  • Exome-Seq: SNPs and small structural variants
  
  We do not offer advanced analysis, but will consider developing pipelines that will be useful to many investigations, e.g., for new techniques.
  
  Charges may include the cost of computing as many of the techniques may require up to a 1000 hours of CPU time."

• Library preparation service ( Material production service )
  

  • Low throughput and high throughput options available
  • Library prep for ChIP-Seq, DNA-Seq, RNA-seq, etc.
  • Many kits available

• Next-generation sequencing data delivery ( Support service )
  

• Public repository submission support service ( Support service )
  

• Sample and library quality assessment service ( Material analysis service )
  

• Single cell genomics services ( Material analysis service )
  

  Services include:
  • capture on C1
  • post capture phase of C1, ether RNA/cDNA or WGA
  • Fluidigm IFC for DNA extraction and WGA
  • Fluidigm IFC for RNA extraction and cDNA

Web Links:

• http://www.med.upenn.edu/cores/NextGenerationSequencingCore.html