Frequently Asked Questions
What services does the Sequencing Facility provide?
Who can order services through the Sequencing Facility?
How do I submit a sequencing proposal?
How do I submit samples for PacBio Sequencing?
What are the quality/quantity requirements for submitted samples for PacBio Sequencing?
What happens after my sample is submitted for PacBio Sequencing?
How do I check the status of my order/sample for PacBio sequencing?
What happens during PacBio library preparation?
What is the difference between CLR and CCS?
What is the estimated output?
What can I sequence on one SMRT Cell?
How can I extract HMW DNA?
Which data will I receive from PacBio sequencing?
Answers
What services does the Sequencing Facility provide?
Please see the services page for a detailed list of projects we support. If your project design is not listed, please contact ccrsfhelp@mail.nih.gov, or the Sequencing Facility Director, to discuss the feasibility of a custom project.
Who can order services through the Sequencing Facility?
All NIH research labs are eligible to order services through the Sequencing Facility.
How do I submit a sequencing proposal?
Please complete a sequencing proposal form at NAS Request , You may also contract ccrsfhelp@mail.nih.gov,to discuss the available platforms and best choice for your project.
How do I submit samples for Pac-Bio sequencing?
Before submitting samples, ensure that the sequencing project has been discussed with the Sequencing Facility team and the NAS request submitted. PacBio service is listed under Sequencing Facility – Pac Bio (CCR)
You may then submit samples by delivering them to ccrsfhelp@mail.nih.gov, at the ATRF Room D3040 (instructions for sample delivery link). Be sure to include a sample manifest with your submission.
What are the quality requirements for submitted samples?
All samples must be sent in a 1.5 ml or 2 ml tubes.
Quality and quantity requirements are listed in the table below:
| Type of Library | Minimum DNA/RNA Quantity Requirement | Recommended DNA/RNA Quantity Requirement | Minimum Concentration Requirement | Quality Requirements |
| WGS | 2 µg | 5 µg | n/a | |
| Amplicons (< 5000 bp) | 100 ng | 300 ng | n/a | OD260/280 1.8-2.0 |
| Amplicons (> 5000 bp) | 200 ng | 400 ng | n/a | OD260/230 1.7-2.2 |
| HLA (Class I) | 250 ng | 1 µg | 20 ng/µl | |
| WTS | 300 ng | 1 µg | 50 ng/µl | RIN ≥ 8.0 |
What happens after my sample is submitted?
Before sequencing, we will perform an internal QC to confirm the information in the sample manifest and notify you if any samples do not meet minimum sequencing requirements. You will then be able to choose whether to resubmit those samples or continue and sequence them at your own risks. You will be notified again when the analysis on each sample is completed and available for download.
How do I check the status of my order/sample for PacBio sequencing?
For information about sample status, please contact us at LongReadInfo@mail.nih.gov , or you can email caroline.fromont@nih.gov.
What happens during PacBio library preparation?
After initial sample QC, we proceed with library preparation. Depending on the project, the samples will be handled differently prior to PacBio library preparation. For amplicons samples, we first perform an AMPure bead clean-up that also allows us to concentrate the samples if necessary. For gDNA samples, we shear the samples to the targeted size depending on the project need, perform an AMPure bead clean-up to concentrate the samples and remove single-stranded overhang. For WTS, we generate cDNA using polydT primers and TSO allowing us to target full length transcripts with a polyA tail. During PacBio library preparation, fragments undergo damage repair, end-repair/A-tailing and adapter ligation. The adapters are hairpin adapters and, ligated to double stranded DNA, they form a circular molecule necessary for PacBio sequencing. Barcoded hairpin adapters are also available if the project require pooling of multiple samples. The libraries are then cleaned using AMPure beads and we perform a QC prior to setting up a sequencing run.
What is the difference between CLR and CCS?
CLR stands for Continuous Long Reads and are produce when the insert size of the library is > 20 kb while CCS stands for Circular Consensus Sequences. CCS are produced when sequencing libraries with insert size shorter than 20 kb. For CCS, the circular template (dsDNA with hairpin adapters) generated during library preparation is read multiple times and produce numerous read passes (subreads). Those subreads are then used to call a consensus sequence and generate highly accurate reads. Four passes of the molecule usually yield Q20 data while 8 passes should yield Q30 data. On the other end, CLR will trade-off accuracy for length as longer inserts will generate less subreads but can sequence reads > 50 bp.
For a quick explanation of SMRT sequencing, please watch the following PacBio video:
PacBio Sequencing – How it Works
On the PacBio website:
What is the estimated output?
Please note that these are estimated output only as both library type and insert size are likely to influence the generated output and it is subject to change. The Sequel II is estimated to produce 3-4.5 million raw reads that may be translated to 200-400 Gb of raw data for CCS libraries and 100-120 Gb of raw data for CLR libraries.
What can I sequence on one SMRT Cell?
According to PacBio, one SMRT cell is enough to sequence a genome up to 2 Gb and a whole transcriptome, detect structural variant in up to 2 samples of ~3 Gb genome, and multiplex numerous amplicons. For variant detection (single nucleotides, indels and structural variants) in a ~ 3 Gb genome, using at least 2 SMRT cells is recommended.
How can I extract HMW DNA?
PacBio has released a list of HMW DNA extraction protocols that can be found at https://extractdnaforpacbio.com/
Which data will I receive from PacBio sequencing?
For further questions please contact us directly CCRSF_IFX@nih.gov.
For ordering and pricing information, head back to Resources.