CHOP Research Institute

Center for Applied Genomics

Today's Research Becomes Tomorrows Cure

Next Generation Sequencing (NGS) at CAG

The goal of our NGS lab is to deliver high quality sequencing services to the science community and our external partners. We are fully equipped with state-of-art technology and experienced staff members. Our lab is integrated with our Biorepository and Bioinformatics group, keeping all stages of the study connected.

Services

CAG offers high quality services for several sequencing applications. CAG-NGS facility can process well-established methodologies or novel in developing by researchers on a design-specific basis.

Project Initiation

CAG's Sequencing Director, Renata Pellegrino, PhD can guide investigators through the experimental design phase by way of a This email address is being protected from spambots. You need JavaScript enabled to view it..
Together with our Bioinformatics team, the CAG Sequencing team can discuss the best approaches and technology for specific experiments.

Instruments

CAG has a full equipped Illumina Sequencing lab, including two Illumina Hiseq2500 SBS v4 instruments and one Illumina MiSeq.

HiSeq Throughput

Our HiSeq v4 reagent kits generate up to 1 terabase (1Tb) of data per 6-day run (up to 500 Gb per flow cell), increasing daily throughput to 167 Gb per day. The new v4 reagents increase the number of clusters by 33% compared to the TruSeq SBS Kit v3, adding additional capacity for counting assays.

KIT NAME OUTPUT MAX
(PER 2-FLOW CELL)
NO. OF READS MAX READ LENGTH TIME
HiSeq SBS V4 Kits Up to 1 Tb Up to 4 billion 2 x 125 bp 6 days

HiSeq Performance Parameters

High Output Run Mode*
HISEQ SBS V4 SPECIFICATIONS TRUSEQ SBS V3
Read length Dual Flow Cell Single Flow Cell Dual Flow Cell Run Time Dual Flow Cell Single Flow Cell Dual Flow Cell Run Time
1×36 128-144 Gb 64-72 Gb 29 hrs 95-105 Gb 47-52 Gb 2 days
2×50 360-400 Gb 180-200 Gb 2.5 days 270-300 Gb 135-150 Gb 5.5 days
2×100 720-800 Gb 360-400 Gb 5 days 540-600 Gb 270-300 Gb 11 days
2×125 900-1 Tb 450-500 Gb 6 days N/A N/A N/A
Reads Passing Filter (8 lanes per flow cell) Up to 4 billion single read or 8 billion paired-end reads Up to 2 billion single read or 4 billion paired-end reads Up to 3 billion single read or 6 billion paired-end reads Up to 1.5 billion single read or 3 billion paired-end reads
Quality Greater than 85% of bases above Q30 at 2×50 bp Greater than 85% of bases above Q30 at 2×50 bp
Greater than 80% of bases above Q30 at 2×100 bp Greater than 80% of bases above Q30 at 2×100 bp
Greater than 80% of bases above Q30 at 2×125 bp
*Install specifications based on Illumina PhiX control library at supported cluster densities (between 610-678 K clusters/mm2 passing filter using TruSeq v3 Kits or 870-930 K clusters/mm2 passing filter using HiSeq v4). Run times for high output mode correspond to sequencing only. Performance may vary based on sample quality, cluster density, and other experimental factors.


Rapid Run Mode*
HISEQ RAPID SBS KIT V2 SPECIFICATIONS
Read length Dual Flow Cell Single Flow Cell Dual Flow Cell Run Time
1×36 18-22 Gb 9-11 Gb 7 hr
2×50 50-60 Gb 25-30 Gb 16 hr
2×100 100-120 Gb 50-60 Gb 27 hr
2×150 150-180 Gb 75-90 Gb 40 hr
2×250 250-300 Gb 125-150 Gb 60 hr
Reads Passing Filter (2 lanes per flow cell) Up to 600 million single read or 1.2 billion paired-end reads Up to 300 million single read or 600 million paired-end reads
Quality Greater than 85% of bases above Q30 at 2×50 bp
Greater than 80% of bases above Q30 at 2×100 bp
Greater than 75% of bases above Q30 at 2×250 bp
*Install specifications based on Illumina PhiX control library at supported cluster densities (between 700-820 K clusters/mm2 passing filter using HiSeq Rapid v2 Kits). Run times for rapid run mode correspond to on-board cluster generation (1.5 hr) and sequencing. Performance may vary based on sample quality, cluster density, and other experimental factors. Early HiSeq 2000 instruments will run slightly slower when upgraded to a HiSeq 2500.

MiSeq Specifications

Cluster Generation and Sequencing
MISEQ REAGENT KIT V2
READ LENGTH TOTAL TIME OUTPUT
1 × 36 bp ~4 hrs 540-610 Mb
2 × 25 bp ~5.5 hrs 750-850 Mb
2 × 150 bp ~24 hrs 4.5-5.1 Gb
2 × 250 bp ~39 hrs 7.5-8.5 Gb
MISEQ REAGENT KIT V3
READ LENGTH TOTAL TIME* OUTPUT
2 × 75 bp ~21 hrs 3.3-3.8 Gb
2 × 300 bp ~56 hrs 13.2-15 Gb


MiSeq Reads Passing Filter
MISEQ REAGENT KIT V2
Single Reads 12-15 M
Paired-End Reads 24-30 M
MISEQ REAGENT KIT V3
Single Reads 22-25 M
Paired-End Reads 44-50 M


MiSeq Quality Scores
MISEQ REAGENT KIT V2
>90% bases higher than Q30 at 1 x 36 bp
>90% bases,higher than Q30 at 2 x 25 bp
>80% bases,higher than Q30 at 2 x 150 bp
>75% bases higher than Q30 at 2 x 250 bp
MISEQ REAGENT KIT V3
>85% bases,higher than Q30 at 2 x 75 bp
>70% bases,higher than Q30 at 2 x 300 bp

General Information

Type of Run – Single Read (SR) or Paired End (PE)
The type of run is related to the application chosen. Single-read runs are characterized by the sequencing instrument reading the fragment/sequence from one end to the other end only. Paired-end runs read from one end to the other end, following another round of reading from the opposite end. In general, single read runs are faster, less expensive, and typically adequate for RNA-Seq or ChIP-Seq. Paired-end runs are more robust in terms of data, provide further positioning information in the genome, being more suitable for de novo genome assembly and resolution of structural re-arrangements such as deletions, insertions and inversions. Paired-end runs are also important for studies involving deep RNAseq (splice variants), epigenetic modifications (methylation) and SNP identification.

Applications Offered

  • Target Sequencing/Custom panels
  • Exome Sequencing
  • Whole Genome Sequencing
  • RNA-Seq

    Novel:
    10X genomics GemCode Technology:
  • Structural Variants
  • Breakpoint Analysis
  • Phased Sequencing

    Infrastructure

    Full automation for library preparation (small, medium and high throughput):
  • 2xHiseq2500 and MiSeq
  • 2 Sciclone G3 NGS Liquid Handling Robots
  • 1 Zephyr NGS Liquid Handling Robot
  • Covaris LE for high throughput shearing
  • Caliper GX electrophoresis
  • Agilent Bioanalizer
  • Agilent Tape Station
  • Qubit, DropSense, SpectraMax, Nanodrop8000, Viia7 qPCR, other liquid handling robots.

    Sample QC and Library Preparation

    Upon receipt of the samples, CAG performs an initial DNA or RNA quality control (QC) assessment to evaluate the concentration and quality for each sample. At this stage, we will have an open channel of communication between the researcher and us to guarantee that all samples reaches the initial strict QC phase before going to library preparation and or sequencing.

    Sample Submission

    General QC Criteria
    DNA RNA
    OD ratios:
    260/280 1.6 - 2.1 (max)
    260/230 above 1.5
    OD ratios:
    260/280 1.8 - 2.2 (max)
    260/230 above 1.5


    Additional QC
    Agarose Gel or Capillary electrophoresis (Bioanalyzer, TapeStation)
    Comments
  • Please provide high quality and non-degraded genomic DNA.
  • Let us know when the samples are special and challenging.
  • Shearing conditions (target enrichment) may change,according to each library preparation
  • CAG will run DNAs on an agarose gel and a Qubit fluorometric analysis to test for DNA quality and quantity.
  • If the samples do not meet our specifications, CAG reserves the right to refrain from sequencing them and will request a new aliquot.


  • Library preparation at CAG
    Minimum DNA Sample Requirements
    Library Type Input Min Volume Max Volume Min Ideal Concentration
    Whole Genome Sequencing, general samples Kapa 500ng 25ul 130ul 25ng/ul
    Whole Genome (special samples) Kapa 20ng 20ul 130ul 3ng/ul
    Whole Exome (Agilent SureSelect XT)* V5 200-500ng 20ul 120ul 10ng/ul
    Whole Exome Agilent SureSelect XT V5 2000ng (2ug) 15ul 120ul 10ng/ul 20-50ng/ul
    Custom Target Enrichment This email address is being protected from spambots. You need JavaScript enabled to view it. This email address is being protected from spambots. You need JavaScript enabled to view it. This email address is being protected from spambots. You need JavaScript enabled to view it.
    Other This email address is being protected from spambots. You need JavaScript enabled to view it. This email address is being protected from spambots. You need JavaScript enabled to view it. This email address is being protected from spambots. You need JavaScript enabled to view it.


    Sequencing only at CAG (library ready)
    Required Amount nmols/L(M)
    Agilent SureSelect protocols (Indexed and Non-Indexed) 10ul 2
    Nimblegen/Kapa solution-based protocols (Indexed and Non-Indexed) This email address is being protected from spambots. You need JavaScript enabled to view it. This email address is being protected from spambots. You need JavaScript enabled to view it.
    Illumina prep protocols This email address is being protected from spambots. You need JavaScript enabled to view it. This email address is being protected from spambots. You need JavaScript enabled to view it.
    Nugen This email address is being protected from spambots. You need JavaScript enabled to view it. This email address is being protected from spambots. You need JavaScript enabled to view it.
    Other This email address is being protected from spambots. You need JavaScript enabled to view it. This email address is being protected from spambots. You need JavaScript enabled to view it.
    QC Method used (qPCR, Bioanalyzer, Qubit)
    Please normalize the samples according to your type of capture/sequencing.
    Do not give the CAG NGS team your entire stock sample.
    Please be aware than any aliquot you provide might be completely used.


    Sample type
    DNA: Genomic Plasmid Cosmid cDNA BAC PCR product Other
    RNA: Total RNA polyA RNA Other
    Sample buffer: IowTE EB Mol grade water Other

    Sample Manifest

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    Contact Details

    For billing information, sample processing and other general questions, you can email Sequencing Director, This email address is being protected from spambots. You need JavaScript enabled to view it..
    You can also contact Renata by phone at (267) 426-0181.