NextGen DNA Sequencing I FAQs   ///  (352) 273-8050   ///    RRID:SCR_019152

Frequently Asked Questions


General FAQs

How do I submit my samples?

In order to submit samples, please first create an account in ICBR CrossLab, or if you are already a customer, please submit a request and obtain a submission ID. Once you have generated a submission ID, please complete a service submission form and include it with your sample at the time of submission. All samples MUST BE submitted in a 1.5-ml tube and labeled clearly with the NS number from your iLab request, and sample name or number consistently with the same identifying information as on the form.

How do I submit a request to iLab?

Please follow the instructions in this document to successfully submit a request to iLab for NextGen DNA sequencing: iLab Service Request Submission Guide.

Can I use Nanodrop concentrations to pool my libraries for sequencing?

It is broadly agreed that for the purpose of NextGen sequencing, quantification should be done using a fluorescence-based method (e.g., picoGreen or Qubit). Nanodrop or any other absorbance-based methods, while useful as an indicator of purity, they tend to overestimate mass. The reason for this is that too many things absorb at 260 nm in addition to RNA and DNA. If you do not have access to a fluorometer, a gel can give an idea of quantity and quality, but fluorescent dyes are a more reliable method for quantifying mass in your sample. The fluorometric methods we recommend (picoGreen or Qubit) utilize dyes that can specifically distinguish double-stranded DNA from RNA or other substances that may absorb at or close to 260 nm. Alternatively, ICBR has a Qubit quantitation service for DNA libraries.

Where do I deliver my samples?

Create a myICBR submission ID, place your 1.5-ml tubes with the completed form in a Ziploc bag and deliver your samples to our staff at ICBR NextGen DNA Lab located at CGRC Building, room 178.  Our lab does not have drop-off freezers.

If the Core performs QC on my samples/libraries, why do I need to bother with providing any data about my sample/library quantity and quality?

Our lab QC services should be considered an independent verification of quality and quantity of submitted material. It is your responsibility to make sure that submitted DNA meets the requirements for the requested services. It only makes sense that you should have a reasonable idea of what you are providing the core facility for analysis. Unfortunately, you will be billed for any QC work that we performed on your samples even if they were determined to be unsuitable for the service(s) that you requested. Please review carefully all information provided under the “Sample Requirements” section.

What type of NextGen DNA Sequencing Services does your facility offer?

ICBR NextGen Lab currently offers library preparation for Illumina, PacBio and Oxford NanoPore. Sequencing services are supported for Illumina, PacBio, and Oxford NanoPore. Our instruments include: Illumina NovaSeq X Plus, Illumina MiSeq, Illumina iSeq, PacBio SEQUEL IIe, ONT PromethION, and ONT MinION Mk1c.

What is the turnaround time for sequencing on any of the instrument platforms?

Our policy is to queue projects on “first-come, first-served” basis. Turnaround time varies depending on the length of the queue at the time of sample submission and on the size of the project. An estimate for project completion time is provided to our clients at the time of sample submission. Sequencing runs on the MiSeq and iSeq for customer-constructed libraries can typically be completed within approximately one week from the time that the samples arrive in the lab. For projects that require library construction by the core, the time of completion will be influenced by the size of the project (i.e., number of libraries.) In our experience, for most projects, this typically adds about one week to the time of completion. For the Novaseq X Plus, with 8 lanes per flow cell, runs can only be set up once we have received enough requests of the same run configuration. This can often take a few weeks for some run formats. The PacBio SEQUEL IIe main sequencing consumable unit is based on 4 SMRT cells runs. So, we need to have at least four samples to set up a run.

Why did my sample fail to sequence?

Sequencing success depends on many factors. Our users must pay close attention to our sample input requirements. These vary depending on the sequencing platform. When a sample does not sequence successfully, we work with our customers to identify possible causes for sample failure. The likelihood of sample failure is rare, but we are unable to guarantee the success of every sample. In the unlikely event, customers will still be expected to pay for requested services, unless we have determined that failure was due to instrument malfunction or acts of unwilling negligence in the core lab while we processed your sample. Please read our customer agreement document carefully.

What type of sequencing data do you provide?

Sequencing data are provided in industry standard formats. For PacBio these include: 1) Bam files containing raw data for the reads; 2) FASTA, FASTQ and CSV files containing filtered subreads and 3) all metadata files associated with the run. For Illumina, we provide the Bcl and FASTQ files.

All data are delivered electronically via Globus.

Our sequencing services do NOT include post-sequencing run data processing or analysis.

Who do I contact and ask questions about my project while it’s being processed?

Once you have a submitted a service request and your samples have been received, your sequencing project is assigned to one of the scientists in our group. The scientist will be the project manager for your samples. This person will serve as your point of contact for any questions related to your project. The most effective way to contact our facility is by email, You can also reach us by phone at 352-273-8050.

Where do I deliver or ship samples to NextGen Sequencing?

You can either bring your samples to our lab or ship them frozen overnight. All samples should be accompanied by a completed service request form. To ship, send via FedEx Overnight using dry-ice boxes. We cannot accept weekend delivery, so please ship early in the week.

Ship to:

ICBR NextGen DNA Sequencing Lab

2033 Mowry Road

CGRC Building, ICBR Room 178

Gainesville, FL 32610.

FAQs: Illumina Sequencing

How can I decide what MiSeq sequencing format I need?

There are a few things to consider before selecting a sequencing format.

  • Five MiSeq kits are available for purchase from Illumina: v2 kits (50-cycles, 300-cycles, 500-cycles) and v3 kits (150-cycles, 600-cycles). The number of cycles refers to the maximum number of bases that can be sequenced with the particular kit. The kit should have enough cycles to sequence read1, index 1, index2 and read2. For example, a 500-cycle kit is used to sequence 250 bp from read 1 and 250 bp from read 2 (aka 2×250) the kit has enough reagents to sequence index 1 and index 2.
  • Number of reads is also determined by the kit format (v2 and v3). MiSeq v2 kits generate 12-15 million single reads and 24-30 million paired-end reads/ MiSeq v3 kits generate 22-25 million single reads and 44-50 million paired-end reads. For example: If your pool has 12 libraries a successful MiSeq run will generate ~2 million single reads per library when using the v3 kit, therefore you should know how many reads are needed per library or sample in order to know which kit to select.
  • The library size is also a very important aspect that will help you choose the most appropriate MiSeq run format. A library that is 650 bp can be sequenced with a 500-cycle kit (2×250) or a 600-cycle kit (2×300). Some researchers prefer to have read 1 and read 2 sequences overlapping by a minimum of 10-bases. Please note that a library size includes the insert (in this case 520 bp) and the adapters (130 bp) for a total of 650 bp. However, the read 1 and read 2 sequences are coming from the insert only.

How can I decide which NovaSeq X Plus sequencing format I need?

  • The number of reads generated by the NovaSeq X Plus is determined by the type of kit that is used for the run. There are three different reagents kits available: 100-cycles, 200-cycles, and 300-cycles. The NovaSeq X Plus’s flow cell has eight lanes that can be loaded with independent samples/projects. Each lane generates ~1.25 billion reads per lane. However, all lanes must have the same format for any given run. The most popular format is 2×150. We can perform runs in any format, for as long as we have sufficient samples for 8 lanes. If your project requires less than eight lanes, we will need to wait until other researchers request runs of the same configuration (format) before we can set up a sequencing run.
  • The nature of the sequencing application and your experimental questions will dictate the length of the reads and the number of reads that you need per sample. Please consult with the Core or with your data analysis counterpart for recommendations.

How much of volume do I need for Illumina sample QC on the TapeStation and the Qubit fluorometer?

If you only require sample or library QC, please contact the Gene Expression & Genotyping Core Lab ( If you need both QC and sequencing services, size-selection, and/or pooling services, please submit directly to the NGS lab. Provide an absolute minimum of 10 ul (5-10 nM by Qubit) per sample to perform these assays. For more information, please contact our lab for more information (

Can I submit individual Illumina libraries for QC before the sequencing run?

Yes, you can bring your individual libraries for QC on Qubit and TapeStation before pooling. We send you the results and you have the option of pooling them in your lab or we can pool them for a fee. We also encourage our clients to do qPCR of the final pool. No separate submission is needed for qPCR quantitation for libraries that are submitted to the NGS lab.

The qPCR is the most reliable method for library quantification because it only quantifies the molecules that have both adapters.

For PCR-free libraries each individual library must be quantified with qPCR. For all other library types, we strongly recommend doing qPCR on the final pool.

FAQs: PacBio Sequencing

What types of projects is the PacBio best for?

The PacBio SMRT sequencing system has unique capabilities that are not available on any DNA sequencing technology. It produces extraordinarily long reads with average polymerase read lengths of 10,000 to 50,000 bp (depending on the type of library), with the longest reads over 100,000 base pairs. Because some of the long reads traverse repetitive or otherwise complex genomic regions, they greatly facilitate de novo assembly processes.  Another unique feature of the PacBio SMRT system is its ability to detect a wide variety of base modification events directly from a standard sequencing run. Single-molecule sequencing of unamplified double-stranded DNA, in conjunction with powerful image processing algorithms, makes it possible to detect strand-specific modifications (e.g., hemimethylation) for analysis of epigenetics phenomena.

Long reads also work very well for sequencing full-length transcripts for isoform analysis (IsoSeq.) Another popular application is sequencing of specific targets that can be generated by either hybridization capture or via long-range PCR.

What’s the first step to start your PacBio sequencing project at the ICBR?

We encourage an initial consultation (meeting between potential users and our Core lab scientists.) This consultation is free of charge. PacBio sequencing is done directly on the native DNA molecule (no amplification.) This puts a “premium” on sample preparation and users need to be aware of the nuances. In addition, our Core lab scientists are in close contact with the larger community of users of this technology and can provide useful information about the latest developments and improvements of the system.

What are the sample requirements for PacBio sequencing?

DNA must be pure (OD260/280 ratio of 1.8 to 2.0; OD260/230 ratio >2). Large-insert libraries require DNA of high integrity (>30 kb). In addition, because there is no amplification, PacBio requires more material than other sequencing systems. Any sample handling that may result in DNA damage (e.g., heat, freezing/thawing, ethidium bromide staining, UV, etc.) will compromise the quality of the sequencing process and thus should be avoided. Required sample amounts for DNA vary from 500 ng to 30 micrograms depending on the desired insert size and the application. RNA (for IsoSeq) must be intact (RIN > 8). A minimum amount of 2 ug of fluorometrically quantitated RNA is required. Please see also our sample requirements document.

What are the specs for PacBio sequencing?

Typical run metrics per library type: PacBio SEQUEL, 20-hr movies  
    Output (Gb) Av. Polymerase read length(bp)  
  Genomic DNA 10-20 15-25  
  IsoSeq 20-40 Gb 20-45  
  Amplicon 20-50 Gb 25-60  


FAQs: 16s Metagenomics

What is 16s metagenomics workflow?

There are several steps involved in 16s metagenomics projects including sample collection, DNA extraction, library preparation, library quantification, sequencing and statistical data analysis.

  1. Sample Collection: A robust sample collection protocol that maintains the integrity of samples is both important and challenging in microbiome studies. The microbial composition of samples may be adversely affected by the collection process. Please visit the following links to find out what’s the best collection method for your samples.
  1. DNA isolation: The challenge here is to use a method that extracts DNA with “similar” efficiency for all microorganisms in order to reveal full diversity in the sample. Please check the following links to find out what extraction kit is best suited for your samples. You can always seek the information from the expert in the field you are working on to see which kit/method they use.
  1. Library Construction (16s amplicon or any other target): ICBR Gene Expression & Genotyping Core currently offers 16s library construction services using either “Quick-16S NGS Library Prep Kit” from Zymo or “Swift Amplicon 16S+ITS Panel” from Swift. If you are constructing your own libraries, we recommend the method described by Illumina and the barcoding reagents provided in the Nextera XT kit (Illumina Inc.) The method consists of two sequential PCR reactions. You will need to order HPLC- purified primers and PCR reagents for the first reaction. The first PCR is identical for every sample. In contrast, the second PCR reaction is unique for every sample. That’s because you need a unique barcode for each sample. You can barcode up to 96 samples to be pooled for a single MiSeq run (2×300 cycles) when using the Nextera XT kit. Other library construction protocols are also accepted. These include, for example, libraries constructed with the 16S Zymo Research kit or custom protocols as described in the literature.
  2. Library quantification, sizing, and pooling: Use QUBIT for quantification and the TapeStation (or Bioanalyzer) for sizing. If you have followed the Illumina recommended method for amplifying variable regions 3 and 4, the size of your library should be ~600 bp). Calculate molarity using Qubit concentration and average size on TapeStation and pool equimolarly.
  3. Sequencing run: For 16s (variable regions 3 and 4), we recommend MiSeq, version 3, 2×300 cycles with 20% PhiX spike-in and 10 pM loading. For a pool of 96 samples, this run format should yield ~100k reads per sample. Of course, it is your choice to request any other run configuration based on the goals of your experiment. Please keep in mind that version 2 configurations (e.g., 2×250 cycles) generate roughly half the number of reads as version 3 reagents (e.g., 2×300 cycles).
    For 16s projects that were submitted for library construction at ICBR GE core, the pooled library will be transferred directly to NextGen core for sequencing run. If you also need support with data analysis, email us at:

    For premade 16s libraries, please submit pool to the ICBR NextGen DNA sequencing core. We feel that it is best practice to request independent library pool evaluation services before sequencing, including quantification of “functional” library molecules by qPCR. You can choose to decline these services, in which case you will need to provide the core with all QC data for your pool, plus detailed conditions for sequencing run set up on the MiSeq. We need at least 15 ul at 10 nM (~3-5 ng/ul). To submit sequencing requests, your lab will need to have a CrossLab account (PI and lab members.) From our website, use your account credentials to login to CrossLab and select UF-ICBR core. Navigate to the NextGen Sequencing core> Illumina sequencing services> MiSeq, etc. to fill out service request form.

  1. Data analysis: You will need to do that on your own using your favorite method or select data analysis services in your submission form. If you decide to request data analysis support, the ICBR Bioinformatics Core can do this for a fee and will contact you to discuss the process. Email us at:

What are the DNA quality requirements?

High sample quality is essential for successful 16s metagenomics experiments. Please ensure that your DNA sample:

  • Is double-stranded. Single-stranded DNA will not ligate to adapters in the library preparation process and can interfere with quantitation.
  • Has undergone a minimum of freeze-thaw cycles
  • Has not been exposed to high temperatures – (> 65oC for 1 hour can cause a detectable decrease in sequence quality), pH extremes (< 6 or > 9)
  • Has not been exposed to intercalating fluorescent dyes or ultraviolet radiation. If purifying DNA from a gel fragment, DO NOT use ethidium bromide for staining. We recommend SYBR Safe DNA Gel Stain with visualization on a blue light box: Even a few seconds of UV irradiation appears to render DNA non-sequenceable.
  • Has an OD260/280 ratio of approximately 1.8 to 2.0 (Nanodrop). Also, the OD260/230 ratio should higher than 2.0.
  • Does not contain insoluble material
  • Does not contain RNA contamination
  • Does not contain chelating agents (i.e. EDTA), divalent metal cations (i.e.,Mg2+), denaturants (Guanidinium salts, Phenol) or detergents (SDS,Triton-X100)
  • Does not contain carryover contamination from the starting organism/tissue (heme, humic acid, polysaccharides, polyphenols, etc.) Some DNA isolation procedures might contain contaminant(s) that can affect the quality of the sequencing run.
  • Is quantified by a fluorometric method (QUBIT or PicoGreen.) Nanodrop (absorbance) is not adequate for quantification, although it does provide valuable information about a sample’s purity.

 How should I quantify the DNA samples?

Use QUBIT (fluorescence-based method.) Nanodrop (absorbance) is not adequate for quantification, although it does provide valuable information about a sample’s purity. Gel electrophoresis (or equivalent TapeStation) may also be important if you’re concerned about the integrity of the samples.

How much DNA is needed for 16s metagenomics library prep?

The standard protocol for Zymo kit requires between 5-20 ng of total DNA. For Swift kit, 1ng is recommended and sufficient yields are attainable from as little as 10pg-50ng, depending on sample type.

Which kits does GE use for my 16s metagenomics project? What is the difference of those kits?

We use either “quick-16S NGS Library Prep Kit” from Zymo or “Swift Amplicon 16S+ITS Panel” from Swift. The Zymo kit uses two primer sets to amplify V1 and V2, or V3 and V4. The Swift kit utilizes five primer sets to amplify and create libraries for all V1-V9 and fungal ITS1 and ITS2 genes in a single primer pool.

Will the GE Core send QC results before starting my library prep?

We will perform a Qubit assay. We also randomly pick 4 samples to run a gDNA TapeStation to assess the integrity of gDNA. If you would like to run TapeStation for all your samples, per sample cost is $7.20. This is not included in the 16s library preparation service fee. The GE core will communicate with customers to let them know whether or not their samples have passed QC.

How do customers submit replacement samples?

If after initial QC, customers need to submit more sample(s) to the core, please upload the sample table in iLab and clearly indicate which samples are being replaced.

Can customers get back the samples?

Yes. Please make a note in the comment box in the iLab submission form that you want leftover samples back upon the completion of the project. We keep customers’ samples up to 6 months. It is the customer’s responsibility to retrieve the samples on time.

How long does the core keep customer’s leftover samples for?

Due to limited space in the GE cores freezer, we only keep customer’s samples for 6 months. If customers would like samples returned to them then they should contact the core to schedule a day and time to come and pick them up.

What is the turnaround time for a 16s metagenomics project?

Turnaround time depends largely on the volume of activity at the time of sample submission and the complexity of the project. Given typical workloads, library construction requires approximately 2-3 weeks and sequencing-analysis often requires an additional 1–2 weeks (when requested.)

FAQs: RNAseq

Which ICBR core conducts RNAseq projects?

RNAseq projects should be submitted in the Gene Expression & Genotyping core, using the ICBR iLab portal. Once libraries have been constructed, the pooled library will be transferred to the NextGen Core for sequencing. No separate request in iLab is necessary if sequencing is requested along with library construction. To start the RNAseq project, please contact or yanp@ufl.edufor more details and for planning your experiment. Our library construction and sequencing services do not include data processing. Data analysis services can be requested separately from

Should customers make an appointment to discuss their RNA-seq project?

We suggest that you schedule a consultation meeting with us before you start your project. This helps us in better understanding the goals of your experiment. Our Bioinformatics experts can give you suggestions about the most appropriate experimental design to achieve your goals.

What is the Illumina RNAseq workflow using NEB reagents?

  1. Start with total RNA. RIN>8 is best but Illumina RNAseq is quite forgiving.
  2. cDNA synthesis:
    • If you are only interested in polyA transcripts, use the poly (A) mRNA magnetic isolation module with oligo(dT) cDNA synthesis primers. Using the NEB poly (A) capture approach precludes the need for depleting ribosomal RNA, which would otherwise represent ~95% of your reads. For RNA fragmentation, the times can vary depending on the RNA quality. High quality RNA for 2×100 sequencing can be done by using the maximum fragmentation time in the manual.
    • If you are interested in all transcripts, including non-poly (A), long non-coding RNA, you would need to start with the RNA ribodepletion kit. NEB has off-the-shelf products for human/mouse/rat ribodepletion. For other species you’d need custom products such as those offered by NuGen (i.e., AnyDeplete) for which the ribosomal depletion happens after cDNA synthesis.
  3. At this point, you’d be ready to attach the adaptors to the ds-cDNA fragments using the Ultra II RNA Library Prep Kit. The NEB manual is self-explanatory. When you get to the final amplification step with barcoded primers, you’ll need to use primers from the NEBNext Multiplex Oligos set (Dual Index primer set 1.) We would recommend using fewer amplification cycles than those recommended by NEB in the manual. If you are starting with one microgram of total RNA, you probably don’t need more than ~7 cycles.
  4. Perform QC on your libraries: Qubit, TapeStation and qPCR. With mass and size information, calculate molarity.
  5. Depending on your familiarity with the library construction protocol, qPCR quantification may be skipped. However, we strongly suggest quantifying the final library pool using qPCR (the Gene Expression core can do it for $40 /sample or pool). qPCR measures the “functional” molecules in the library (i.e., molecules that contain full p5 and p7 adaptors).
  6. Normalize concentrations and pool equimolarly.
  7. The Gene Expression core will pass the pooled library to the NextGen for sequencing. Illumina recommends ~50 million reads per sample. Since one lane on the HiSeq produces ~300 million reads, you can do 6 samples per lane (barcoded.) However, you can multiplex numerous samples per lane, depending on your experimental goals.
  8. For pre-constructed RNAseq libraries (i.e., libraries that have NOT been made in the Gene Expression Core, please submit directly to NGS core at least 10 ul at 5-10 nM of your pool for sequencing.

How much RNA needed for RNASeq library prep?

Library complexity is very important in an RNA-seq experiment. If the starting RNA is limited, amplification artifacts are more likely to occur which affect the interpretation of the results. The standard protocol for library construction requires between 100 ng and 1 μg of total RNA. There are kits available for ultra-low RNA input that start with as little is 10 pg-10ng of RNA; however, the reproducibility increases considerably when starting with at least 1-2 ng. If possible, 1 to 1.5 ug of total RNA is preferred for sample QC and library prep.

What are the RNA quality requirements?

High sample quality is essential for successful RNA-Seq experiments. Customer should do initial QC of total RNA sample with a NanoDrop. The 260/280 and 260/230 ratios need to be greater than 1.8. More accurate mass measurements are accomplished with the QUBIT (fluorescence-based) method for RNA. To determine total RNA quality ,we use Agilent Bioanalzyer or TapeStation. 7-10 ranges of RIN (>7) of samples are highly recommended by Kit.

What if customers do not have 50 ng input RNA? Does GE offer RNA library prep services?

If a customer has less than 50ng of input RNA, we offer an option for RNA amplification using Clontech’s SMART-seq v4 Ultra Low Input RNA Kit to create amplified cDNA, followed by library preparation using Illumina’s Nextera XT kit. The minimum input required for this kit is 10pg, but >200pg is preferred.

Will the GE Core send QC results before they start my library prep?

Usually ‘Yes’. The GE core will upload QC data to iLab which will contain RIN scores of customer’s RNA samples (when applicable) along with concentration. If the quality and quantity of customer’s samples do not meet the requirements for the library prep, we will let customer know. We will also provide suggestions for possible options to move forward with the project.

Is it OK to isolate samples in batches?

It is best if you can isolate samples at once, but it is common practice that researchers collect/extract RNAs at different times using the same protocol.

Can customers get back the samples?

Yes. Please make a note in the comment box in iLab submission form that you want the leftover samples back upon the completion of the project. We keep customer’s samples up to six months. It is the customer’s responsibility to retrieve the samples on time.

How long does the core keep customer’s leftover samples for?

Due to limited space in the GE cores freezer we only keep customer’s samples for six months. If customer would like samples returned to them then they should contact the core to schedule a day and time to come and pick them up.

What is the turnaround time for an RNA-seq project?

Turnaround time depends largely on the volume of activity at the time of sample submission and the complexity of the project. Given typical workloads, library construction requires approximately 2-3 weeks. If data analysis service is requested, this would typically increase time by an additional 1-2 weeks.

How should I design my RNA-seq experiment?

You can schedule a consultation meeting by contacting or We will schedule a meeting with the participation of members from the Gene Expression, NextGen and Bioinformatics cores.

Which strand is sequenced for my strand-specific RNA-seq data?

When using paired-end sequencing, the forward read of the resulting sequencing data represents the “anti-sense strand” and the reverse read the “sense strand” of the genes.

How do customers submit replacement samples?

If after initial QC, customers need to submit more sample(s) to the core, please upload the sample table in iLab and clearly indicate which samples are being replaced.

Does GE accept FFPE RNA for RNA-seq?

For degraded and FFPE RNA, we offer the TruSeq Stranded Total RNA with Ribo-Zero Gold prep. This uses Ribo-Zero technology to deplete the rRNA (both cytoplasmic and mitochondrial), as the normal protocol will not work due to loss of the poly-A tails. We still require a Bioanalyzer report. We accept RINs of <7 for this protocol but cannot guarantee the quality of sequencing results as the RNA integrity is so variable in FFPE.