FAQ

Answers for General Questions

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. Labs outside of CCR and NIAID will have overhead charges added.

How do I submit a sequencing request?

Please complete a sequencing proposal form at NAS request. There will be an option for Illumina, which you should select for all short read and single-cell projects, and Long Read, which you should select for all PacBio, ONT and Bionano projects. You may also contact ccrsfhelp@mail.nih.gov to discuss the available platforms and best choice for your project.

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.

Answers for Illumina Questions

How do I submit samples for Illumina sequencing?

Before submitting samples, ensure that the sequencing project has been discussed with the Sequencing Facility team and the NAS request submitted. Illumina service is listed under Sequencing Facility –Illumina (CCR).

You may then submit samples by delivering them at the ATRF Room D3040 (instructions for sample delivery. Be sure to include a sample manifest form with your submission as well as to send an electronic version of the form to Jyoti Shetty prior to sending your samples.

What are the requirements for submitted samples for Illumina sequencing?

Sample Quantity/Quality Requirements and Recommendations:

All samples are shipped in dry ice and the individual (1.5-2 ml) tubes are labeled clearly

You can use any extraction protocol as long as the DNA/RNA samples meet our sample requirements.

Here are the requirements for ATAC seq from frozen cells:

• Bulk ATAC-seq requires each sample to be present as a replicate. Triplicates are better.
• Cells for ATAC-seq should be cryopreserved at high viability. Please ensure that the cells are cryopreserved properly in freezing medium.
• Please send a minimum of 2 million cells per sample.
• Please ship the cells in dry ice.

Answers for PacBio Questions

How do I submit samples for PacBio 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 – Long Read Technology (CCR)

You may then submit samples by delivering them at the ATRF Room D3040 (instructions for sample delivery). Be sure to include a sample manifest form with your submission as well as to send an electronic version of the form to caroline.fromont@nih.gov prior to sending your samples.

What are the requirements for submitted samples for PacBio sequencing?

All samples must be sent in a 1.5 ml or 2 ml tubes.

Some requirements might be project dependent such as input of DNA if multiplexing or sequencing on multiple SMRTcells. Please contact us for more details.

Quality and quantity requirements are listed in the table below:

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 and perform an AMPure bead clean-up to concentrate the samples. 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 requires 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 are PacBio HiFi and CCS reads?

CCS stands for Circular Consensus Sequences. CCS are produced for sequencing libraries with insert size shorter than 25 kb. For CCS, the circular template (dsDNA with hairpin adapters) generated during library preparation is read multiple times and produces 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. HiFi reads are CCS reads with > Q20.

For a quick explanation of SMRT sequencing, please watch the following PacBio video https://youtu.be/_lD8JyAbwEo

On the PacBio website: https://www.pacb.com/technology/hifi-sequencing/

What is the estimated output for PacBio sequencing?

Please note that these are estimates only as both library type and insert size are going to influence the output and it is subject to change. The Sequel II is estimated to produce 3-4.5 million raw reads. For RNA iso-seq libraries you can expect to get 3-4 million CCS reads. For WGS libraries, you can expect to get about 20-30 Gb of HiFi reads.

What can I sequence on one SMRT Cell 8M?

According to PacBio, one SMRT cell is enough to sequence a genome up to 2 Gb and a whole transcriptome, detect structural variants 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.

See “What can you do with one SMRT cell?” for more information.

How can I extract HMW DNA?

PacBio has released a list of HMW DNA extraction protocols and QC methods that can be found at DNA preparation technical note

How can I perform target enrichment for PacBio Iso-seq?

If you are interested in long read isoform sequencing but are focused on only one or a few genes you may consider a target enrichment protocol. This protocol relies on hybridization of biotinylated probes to your cDNA target of interest and subsequent pulldown with streptavidin beads. The enriched cDNA is then amplified and prepared for PacBio sequencing. To design probes for your project please contact IDT at NGSDesign@idtdna.com or fill out a probe design request at https://go.idtdna.com/Request-consult-NGS-xGen-Custom-Hyb-Panel. To complete your sequencing request you will need to submit your probe panel in addition to your RNA samples. Please contact us for more details.

Answers for Oxford Nanopore Technologies (ONT) Questions

How do I submit samples for ONT sequencing?

Before submitting samples, ensure that the sequencing project has been discussed with the Sequencing Facility team and the NAS request submitted. ONT service is listed under Sequencing Facility – Long Read Technology (CCR)

You may then submit samples by delivering them at the ATRF Room D3040 (instructions for sample delivery). Be sure to include a sample manifest form with your submission as well as to send an electronic version of the form to Juanma Caravaca prior to sending your samples.

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.

What are the requirements for submitted samples for ONT sequencing?

All samples, except ONT Ultralong, must be sent in 1.5 or 2 mL tubes in dry ice. For ONT Ultralong, send the cells as a frozen pellet or cryopreserved vial in dry ice.

Quality and quantity requirements are listed in the table below:

How can I extract HMW DNA?

We recommend the HMW Circulomics kit (NB-900-001-01) for DNA extraction.

What is the estimated output for ONT sequencing?

• GridION: up to 50 GB per flow cell
• PromethION 2 Solo: up to 200 GB per flow cell

Answers for Bionano Questions

How do I submit samples for Bionano optical mapping?

Before submitting samples, ensure that the sequencing project has been discussed with the Sequencing Facility team and the NAS request submitted. Bionano service is listed under Sequencing Facility – Long Read Technology (CCR)

You may then submit samples by delivering them at the ATRF Room D3040 (instructions for sample delivery). Be sure to include a sample manifest form with your submission as well as to send an electronic version of the form to Juanma Caravaca prior to sending your samples.

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.

What are the requirements for submitted samples for Bionano optical mapping?

• Please send the cells as a frozen pellet (1.5-2 million cells/sample).
• Please use the Bionano DNA stabilizer buffer to prepare frozen pellets as specified in the protocol of Preparing Frozen Cell Pellets Recommended.
• Please ship the cells in dry ice.

What is the recommended coverage for Bionano optical mapping?

Answers for Single Cell Questions

What is sample processing workflow for single cell projects?

Upon receiving single cell suspension, we check for the quality of cells and wash the cells a couple of times with PBS+BSA before loading it onto a microfluidic chip for the capture of single cells in a nanoliter size droplets along with the barcoded beads. RT takes place inside the droplet and then we break the droplets and PCR amplify the cDNA in bulk. We then purify the cDNA and check the quality. Generally, the quality of cDNA correlates very well with the final sequencing results. We then make Illumina compatible libraries from these cDNAs and sequence it on sequencer.

What is Single Cell RNA-Seq?

Single-Cell RNA-Seq provides transcriptional profiling of thousands of individual cells. This level of throughput analysis enables researchers to understand at the single-cell level what genes are expressed, in what quantities, and how they differ across thousands of cells within a heterogeneous sample.

Do dead cells impact the data quality?

10X Genomics Single Cell Protocols require suspensions of viable (90% optimal, 70-90% acceptable), single cells as input. Dead cells easily lyse, resulting in the release of ambient RNA. This cell-free RNA can contribute to the background noise of the assay and will compromise the quality of single cell data.

Clusters of dying cells typically have relatively higher levels of mitochondrial expression, lower gene counts, and more ambiguous cell type identification scores that equally or comparably match multiple major cell types.

How many cells do I need to provide?

We recommend > 1×10⁶ cells/mL – minimum 200,000 cells/mL to load 10K live cells for non-hashing experiments and 20K-30K live cells for cell hashing experiments.

How many cells can I expect to get information for?

The capture rate of 10X is approximately 60%, depending on cell type and cell quality. When you load 10K (70-90% live) cells, you will capture around 6K cells.

How many reads do I need for my experiment?

We aim to provide 20K-50K reads/cell for gene expression libraries, 10K reads/cell for V(D)J enriched samples, 5K-10K reads/cell for CITE-Seq libraries, 50K reads/cell for single cell ATAC libraries, 20K-50K reads/cell for single cell multiome libraries as recommended by 10X genomics.

What 10X applications do you support?

We support following 10X assays: single cell gene expression (3’ and 5’), single cell immune profiling (V(D)J, TCR, BCR), Single cell multiome ATAC+Gene expression, Single Cell ATAC. We also support Mission Bio Tapestri single cell targeted DNA application.

What buffers should I use to resuspend my cells on the day of submission?

10X recommended to use 1X PBS (calcium and magnesium-free) containing 0.04% weight/volume BSA (400 μg/ml) for washing and resuspension. It is also possible to use most cell culture media with up to 10% FBS or up to 2% BSA to maintain cell health with little to no adverse downstream effects. Media should not contain excessive amounts of EDTA (> 0.1mM), or magnesium (> 3mM) as those components will inhibit the reverse transcription reaction. Any surfactants (Tween-20, etc.) should also be avoided as they may interfere with GEM generation.

How should I prepare and send my samples?

We accept fresh and cryopreserved cells. Please use 10X Genomics recommended or supported cryopreservation protocols for your cells, and follow the 10X Genomics full cell preparation guide.

Fresh samples need to be at CCR_SF within 1-1.5 hour after dissociation. Fresh samples need to be loaded onto the 10X machine as soon as possible after dissociation. Please bring your samples before 2pm!

• Cell parameters: Recommended cell number: > 1×10⁶ cells/mL. Minimum 200,000 cells/mL
• Viability: Recommended > 90%. Acceptable 70%-90%.
• Container: Please use 15mL conical Falcon tubes or 2 mL Eppendorf tubes
• Medium: You can hand your dissociated cells over in cell culture medium (up to 10% FBS or up to 2% BSA) or in 1X PBS/0.004% BSA.
• abeling: Please label tubes clearly and use permanent markers or labels. Always label your tubes on the lids and the side. Please use short unambiguous names (e.g., CTRL, IFN1).
• Temperature: Please deliver your fresh cells on ice. Please ship your cryopreserved samples on dry ice.

What is the cost per sample?

All the information about pricing is listed on our website.

What is included in the price?

We provide full service which includes administrative services, consultations, advice on experimental design, 10X genomics reagents, sample QC prior to loading, cDNA QC, post library generation QC, primary bioinformatic analysis (using the Cell Ranger pipeline).

What is the Turn Around Time for your single cell core?

It takes about 6-8 weeks from the time the sample is submitted, to data delivery after running the CellRanger pipeline. Turn around time can increase for large projects (> 48 samples) and close to the end of the fiscal year.

How should I schedule the experiment?

Please email our scientific team. They will reply you and may arrange a short meeting to discuss the project. The scheduling should be at least 2 weeks in advance. When your samples are ready, you will need to submit a NAS request listed under Sequencing Facility, Illumina (CCR). Please fill out a sample manifest form and send back to us.