Sigt

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 Illumina HiSeq2500

 

Lane Output:

Run Type

High Output v3*

(per lane)

Rapid Run v1*

(per lane)

High Output v4

(per lane)

Rapid Run v2

(per lane)

Single End 180M reads 150M reads 250M reads 200M reads
Pair End 360M reads 300M reads 500M reads 400M reads

Lane output is approximate and based on SBS v3 or v4 chemisty with standard TruSeq libraries.

 

Supported Read Length (GRC):

High Output v3*

(per lane)

Rapid Run v1*

(per lane)

High Output v4

(per lane)

Rapid Run v2

(per lane)

1x50 1x50 1x50 1x50
1x100 1x100 1x100 1x100
2x50 1x150 2x50 1x150
2x100 2x50 2x125 2x50
  2x100   2x100
  2x150   2x150
      2x250
       
       

*High output v3 and rapid v1 run types will be restricted to legacy projects only, unless specifically requested by investigator.

HS Specifications

 http://www.illumina.com/systems/hiseq_2500_1500/performance_specifications.ilmn

 

 

Recommended lane usage and output:

Application
Read Length

# Samples per lane

(Max.)

Reads per sample

(Raw)

mRNA-seq
1x100
10
20 million
Total RNA-seq
1x100 or 2x125
5
40 million
Small RNA-seq
1x50
24
10 million
Low-input RNA-seq
1x100
8
20 million
Chip-seq
1x100
15
15 million
Whole Genome (10x)
2x125
2
125 million pairs
Whole Genome (4x)
2x125
5
50 million pairs
Whole genome Bisulfite-seq (10x)
2x125
2
125 million pairs
Whole Exome Sequencing (100x)
2x125
5
50 million pairs
Whole Exome Sequencing (100x)
2x125
5
50 million pairs

**The above recommendations are based on Human/Mouse/Rat genomes for simplicity and empirical results from GRC experience.  Other organisms may require different sequencing depths (more or less samples per lane).  The amount of sequencing performed and the type (single vs pair) is organism and application specific.  These recommendations are guidelines and by no means meant to be strict cutoffs.  Internal GRC data and cumulative data analysis over the past 2 years formed the basis for these suggestions with the sole purpose to help investigators select the most appropriate experiment type for their reasearch goals.

 

Applications (HiSeq2500)

 

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
      • Whole Genome (WGBS)
      • Reduced Representation (RRBS)
      • Enhanced Reduced Representation (ERRBS)
    • 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 >500bp
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:

 

cBot

  • Cluster amplification of sample library

 

MiSeq

Illumina MiSeq

 

MiSeq Specifications

http://www.illumina.com/systems/miseq/performance_specifications.ilmn

 

 

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.