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General Questions
Sample Preparation
Order & Processing
General Questions
1. What is the difference between resequencing and de novo sequencing and assembly?
Resequencing is typically performed when a reference genome sequence is available. Sequencing reads are aligned back to the reference to determine the location in the genome the specific read best matches. Resequencing is often applied to variant detection (single nucleotide polymorphisms, small insertions/deletions, structural variants, copy number variation) and derivatives thereof—such as tumor vs normal comparison, population genetic analysis, Mendelian disease analysis, and trio sequencing.
De novo sequencing and assembly is typically applied to organisms where no reference genome is available or the available reference is of poor quality. Genomes that have not been sequenced before must be assembled via a de novo approach following sequencing. This assembly can then be used for additional analyses and the basis for future resequencing projects.
2. Which sequencing platform should I choose?
The answer is highly dependent on many factors, including the goals of your experiment, the organism of interest, and the amount of material available.
Most resequencing applications are well suited for Illumina platforms. Illumina sequencing produces large numbers of short reads (<300 bp) at high quality allowing for robust variant detection. Illumina datasets are widely applied to genomic studies of viruses, bacteria, mammals, and plants with a wide range of input materials including low input DNA, FFPE samples, and circulating cell free DNA.
However, certain analyses are improved by very long sequencing reads (several kilobases) generated by the PacBio platform. These long reads are able to span large repeat regions, enabling near complete de novo assembly of small to medium sized genomes, improved assembly of large complex genomes (e.g. plants and animals), and more sensitive detection of large structural variants. Long-read sequencing is ideal for complex genomes that could be highly repetitive in nature and/or have GC extremes.
3. How much coverage do I need?
The answer is highly dependent on many factors, including the goals of your experiment, the organism of interest, and the amount of material available.
Below are some generalized recommendations for short-read whole genome sequencing using Illumina platforms:
- Germline/frequent variant analysis: 20-50x
- Somatic/rare variants: 100-1000x
- Tumor vs Normal: ≥60x tumor, ≥30x normal
- Population studies: 20-50x
- De novo assembly: 100-1000x
Below are some generalized recommendations for long-read whole genome sequencing using PacBio platforms:
- Gap filling and scaffolding: 10x
- Large structural variant detection: 10x
- Germline/frequent variant analysis: 20-50x
- De novo assembly: 50-100x
4. How do I calculate coverage?
Coverage is a multiplier based on the total size of the genome (see below). For humans, 30x coverage can be achieved with 600 million reads of 150 bp (or 300M paired-end reads).
Coverage = (read length) x (total number of reads) / (genome size)
Example: 30x = (150 bp/read) x (600 x 106 reads) / (3 x 109 bp)
5. What kit or library preparation protocol is used for short-read whole genome sequencing?
For resequencing of individual human genomes on Illumina platforms, our standard DNA library preparation workflow uses an Illumina-compatible PCR-based library preparation kit. Please contact our NGS team for further questions on library preparation.
6. What bioinformatics analysis does GENEWIZ offer for whole genome sequencing?
For resequencing of individual human genomes on Illumina platforms, our Standard DNA Analysis Package includes SNP/INDEL detection, structural variant analysis, and copy number variation detection. We also offer tumor vs normal variant analysis, joint-genotyping, and population genotyping at an additional cost. Larger structural variants can be better resolved with low coverage PacBio sequencing.
For sequencing of non-human genomes on Illumina platforms, our Standard DNA Analysis Package includes mapping, SNP/INDEL detection, and structural variant analysis. We also offer CNV discovery at an additional cost.
We also provide de novo assembly. Complete to near-complete sequence assemblies can be obtained for most genomes.
7. What read length does GENEWIZ typically obtain on the PacBio Sequel?
We typically achieve average read lengths of 10-15 kb and maximum read lengths of 60 kb. However, many factors influence the results of a particular project. Please contact us for further questions.
8. What are typical data outputs for the PacBio Sequel?
We typically obtain ~8 Gb of raw data per SMRT cell; however, this depends on many factors. Please contact us for further questions.
9. How do I contact GENEWIZ for a technical consultation about my project?
Navigate to the bottom of the page for our contact details. GENEWIZ's NGS Team is composed of Ph.D. scientists who can help you optimize your project design and provide consultation.
10. Can you recommend the best data delivery option?
Yes, we can recommend the best data delivery option based on the platform and project details. Options include:
- Secure File Transfer Protocol (sFTP)
- Customer cloud account
- Hard drive (additional charges may apply)
11.How will my data be delivered?
If your results are being shipped to you via hard drive, you can track its shipment using the tracking number provided to you in the delivery email sent from GENEWIZ.
If your results are being delivered via another data delivery method, such as AWS S3, Aspera, or other cloud-based methods, please refer to the delivery email sent from GENEWIZ for detailed information on the transfer, and consult with your IT department to ensure compliance with your institution’s policies.
If your results are being sent via a secure File Transfer Protocol (sFTP), please refer to GENEWIZ’s sFTP Data Download Guide for instructions on how to download your data and troubleshooting tips.
12. What is the price for Whole Genome Sequencing?
Please submit a quote request to receiving accurate pricing information, as the cost depends on the details of the project.
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Sample Preparation
13. How should I prepare and send my samples?
View our Sample Submission Guidelines for instructions on preparing and sending your samples for whole genome sequencing. Ship samples directly to our facility. Use the shipping address listed on the order receipt.
14. What happens if my samples do not meet your starting material requirements?
Please contact us.
15. How do I properly prepare high molecular weight (HWM) DNA samples for long-read sequencing?
Please refer to our HMW DNA Best Practices for guidelines on extracting and handing these samples.
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Order & Processing
16. How do I request a quote?
For a quick tutorial, watch the video below.
- For short-read (Illumina) whole genome sequencing, select the Whole Genome Sequencing icon.
- For long-read (PacBio) whole genome sequencing, select the PacBio Services icon.
- For genome phasing using the 10x Chromium platform, select the Custom icon.
17. How do I confirm a quote?
For a quick tutorial, watch the video below.
18. How do I submit the GENEWIZ sample form (to provide detailed information about my samples)?
For a quick tutorial, watch the video below.
19. How can I monitor the progress of my project?
The status of your order, including an estimated date of delivery, can be viewed anytime through your GENEWIZ account. Visit the Order Summary page of your project to find the current order status.
20. How long do you hold samples?
We hold samples for 1 year after project completion. Please contact us if you would like samples retained for longer or shipped back to you.
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