This service is performed using state-of-the-art Illumina® NextSeq NGS platform, and includes the following components:
- Construct an NGS library, including linearization of circular DNA and/or template amplification, with QC procedure at each step;
- Sequence the library primer-free with comprehensive coverage at high depth and accuracy;
- Align the reads against a reference sequence, or assemble the reads into a larger contig
In most cases, sequencing data and analysis results are delivered within 2 weeks of sample receipt.
ORDER NOWWorkflow Overview
- We receive your sample and notify you of sample receipt.
- Upon request, sample DNA template is amplified and QC’ed by Qubit fluorometry.
- Sample DNA is used to construct NGS library, and library is QC’ed by 2100 Bioanalyzer electrophoresis.
- NGS library pool is size selected and QC’ed by Qubit fluorometry and 2100 Bioanalyzer electrophoresis.
- Compatible NGS libraries are sequenced by Illumina NextSeq platform.
Bioinformatics Analysis
Standard types of bioinformatics analysis are included with High Throughput NGS:
Map to Reference
Sequenced NGS reads are aligned to the reference sequence to detect genetic variations (SNVs, InDels, CNVs) and generate a consensus sequence.
Application: Confirming sample sequence and detecting sequence deviations
Deliverables: Full project report; NGS sequencing QC report; FASTA-format consensus sequence of sample reads; variant report.
De novo Assembly
Sequenced NGS reads are constructed to a larger, scaffolded set of contigs, representative of the original sequence, without any reference.
Application: Obtaining sample sequence without prior knowledge
Deliverables: Full project report; NGS sequencing QC report; FASTA-format best contig assembly from sample reads.
Note: De novo assembly is not recommended for episome analysis with bacterial culture or colony sample types, due to significant bacterial gDNA contamination. For bacterial genomes, de novo assembly may be incomplete and contain multiple shorter contigs.
Custom Analysis
More complex bioinformatics analysis for various research purposes, such as comparative genomic and transcriptomic analyses, methylation analysis, small RNA analysis, and cancer NGS data, can be arranged on a project-specific basis.
For any custom project/analysis request, please contact us for a quote.
REQUEST A QUOTEHigh Throughput NGS vs Sanger Sequencing
Unlike traditional Sanger sequencing, which relies on primers to evaluate a specific target region, High Throughput NGS service sequences the entire episome or PCR product molecule with high coverage depth and accuracy:
- High Throughput NGS yields reads with quality of Q30 or higher (>99.9% accuracy)
- High Throughput NGS yields coverage of greater than 500X average depth over entire episome sequence
- High Throughput NGS is able to reveal base deviations and small indels, not detectable with Sanger, at higher sensitivity and lower limit of detection
- High Throughput NGS is more cost-effective for medium to large targets longer than 750 bp
- High Throughput NGS is the practical solution for bacterial whole genome sequencing (WGS)
Learn more about ACGT Sanger Sequencing service.
Sample Preparation
ACGT accepts the following sample types for High Throughput NGS:
Purified DNA: Please provide at least 10 µL, with OD260/280 >1.6 and <2.2, and all samples normalized to 3 ng/µL.
ACGT recommends DNA quantification using fluorometry. Quantification using Nanodrop is discouraged.
Note: Data quality of samples not within standard cannot be guaranteed.
Bacterial Colony: Please provide sample colonies on agar plates, with 1 plate/sample.
Bacterial Culture: Please provide sample in tubes, colonies, or plates, with at least 0.2 mL per sample.
For bacterial colony and bacterial culture samples, additional $25 per sample applies for DNA template amplification ($50 per sample for whole genome sequencing).
ACGT accepts the following target types for High Throughput NGS:
Episomes: Plasmids, cosmidcs, phagemids, BACs
PCR Products: Longer than 500bp
Bacterial WGS: Complete bacterial genomes
Sample Shipping
For accurate and timely sample processing and data delivery, please make sure your samples EXACTLY MATCH the information in your order details. Discrepancies may delay or prevent sample processing.
STEP 1: Label your samples
Clearly label tubes with BLACK permanent marker or printing labels exactly as in your order details. Label plates on the sides. If using tubes with screw-on-caps, label both the tube and caps.
Use BLACK markers only. Colored markers may smear and become illegible.
Use simple and short IDs.
STEP 2: Package your samples
All samples should be packaged securely in a ziploc bag within a shipping package, with adequate padding to prevent damage during shipment. Refer to section below for your specific sample container instructions.
Please include a hard copy of your order confirmation page, which may be found in My Account > My Orders (login required).
2. DNA samples in tubes or as a pellet may be shipped at room temperature for overnight delivery. Dry ice or wet ice is not required.
2. ACGT recommends freezing your plate samples and shipping with dry ice. You may pack the plates (while still frozen on dry ice) in a Styrofoam box. Please ensure there is a sufficiency of dry ice on the top, bottom, and on both sides of the plate(s).
3. If shipping more than one plate, wrap plates in aluminum foil and/or vinyl tape (not lab tape) to avoid shifting during shipment. Lab tape tends to become brittle and rip apart when exposed to dry ice.
2. Pack agar plates in the shipping container with the lid side down. Pack with a protective bubble pad.
3. Plates may be shipped at room temperature if not overgrown.
2. Once the plates have cooled, pack agar lawns in a Styrofoam cooler with the lid side down.
3. Pack ice packs in the cooler. Do not place ice packs directly on plates as condensation may leak into the plates.
4. On the container, clearly indicate “THIS END UP” with arrows for proper handling and delivery.
STEP 3: Ship your samples
ACGT Dropbox is the fastest, safest, and most convenient option. Simply place your order in any ACGT Dropbox near you, and enjoy free overnight shipping. Please refer to the nationwide ACGT Dropbox Map below for locations and pick up times.
If there is no ACGT Dropbox near you, please ship your samples to:
ACGT
Attn: MB Group
35 Waltz Drive
Wheeling, IL 60090
It is recommended that samples are shipped by FedEx, though ACGT accepts samples from the U.S. Postal Service and all other courier delivery services.
You will be notified by email once ACGT receives your samples. Once received, please allow 2-3 weeks for standard NGS order processing.
ORDER NOWACGT Dropbox reduces our impact on the environment by combining samples into a single shipment.
Please contact us if you are interested in an ACGT Dropbox at your institution, or enrolling in our courier program!
Contact us at [email protected] for any questions, and we will respond within 1-3 business days. For any custom project/analysis request, please contact us for a quote.
REQUEST A QUOTE