Next Generation Sequencing (NGS)

 

At the URGRC, we utilize Illumina's sequencing by synthesis (SBS) technology.  Illumina is currently the most successful and widely used NGS platform.

Instrumentation

Illumina HiSeq2500 Sequencer

  • 600 GB data output
  • On board pair end turnaround
  • High output (2 flow cells)
  • 180 - 360 million reads per lane of flow cell (single vs. pair end)
    • 6 billion reads per full run of 2 flow cells
  • Current read lengths:
    • 1x100, 1x150 (Single End Read)
      • ~7 day run
    • 2x100, 2x150 (Pair End Read)
      • ~12 day run

cBot

  • Cluster amplification of sample library

 

MiSeq

  • up to 15Gb data output in 65 hours of run time
  • On board cluster generation and pair end turnaround
  • Version 2 (2x250) and version 3 (2x300) reagent kits
  • Applications include:
    • Amplicon sequencing
      • 16s (microbiome)
      • targeted/custom panels
        • Rep-seq
        • cancer panels
    • Small genome sequencing
      • Bacteria, viruses, etc...

Applications

 

Transcriptomics

 

Library Preparation
Total RNA Input
Target capture
Application
TruSeq mRNA-seq 100ng Poly A RNA mRNA detection and differential expression, splice variants, rare transcripts, complementary SNPs
TruSeq Stranded mRNA-seq 100ng Poly A RNA mRNA detection and differential expression, splice variants, rare transcripts, complementary SNPs, captures both coding and non-coding transcripts that are poly adenylated, provides strand information, gene fusions
TruSeq Total stranded RNA-seq
(Human, Mouse, Rat)
100ng Ribosomal RNA depletion (RiboZero) coding and multiple forms of non-coding RNA, mRNA detection and differential expression, splice variants, rare transcripts, complementary SNPs, gene fusions, lincRNAs, works well for low quality RNA samples and FFPE samples
Low input RNA-seq
(Clontech SMARTer Technology)
1ng Poly A RNA mRNA detection and differential expression, splice variants, rare transcripts, complementary SNPs
TruSeq Small RNA-seq 1ug Directional adaptor ligation takes advantage of 5` and 3` modifications of small RNA small regulatory RNAs, including micro RNA

 

Gene Regulation and Epigenomics

 

    • ChIP Sequencing
    • Bisulphite Sequencing
    • MeDIP Sequencing

Genomics

 

Library Preparation
DNA Input
Target capture
Application
PCR Free DNA 1-2ug DNA Whole genome sequencing, SNP/InDel identification, high GC-rich regions, promoters, and repetitive content, reduced library bias and gaps
TruSeq Nano DNA 100-200ng DNA Whole genome sequencing, SNP/InDel identification
Nextera XT 1ng DNA Small genomes, amplicons >300bp,
Nextera 50ng DNA Whole genome sequencing, SNP/InDel identification
Nextera Mate-Pair 1-4ug DNA Whole genome sequencing, De Novo sequencing, genome assemblies
Nextera Rapid Whole Exome 50ng DNA exome sequencing, 37Mb or 62Mb (expanded) target baits, custom targets also available
Agilent SureSelect Whole Exome 4ug DNA exome sequencing, 62Mb+UTRs (version 5), other options available, custom targets also available

 

Contact us for custom applications.

For Illumina NGS library preparation we utilize kit based workflows supplied by:

Data Analysis

The URGRC currently has 2 bioinformaticians on staff with experience in NGS data analysis methods and workflows.  For more information please contact us.