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
Novel:
10X genomics GemCode Technology:
Infrastructure
Full automation for library preparation (small, medium and high throughput):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 |
| 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
This email address is being protected from spambots. You need JavaScript enabled to view it.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.
