Illumina Sequencing

Illumina sequencers deliver the most flexible and longest available reads of the shorter-read-length platforms, crossing important length thresholds to facilitate many genomic applications.

Sequencing on an Illumina sequencer can be done by generating data from one end (single-end reads=SE) of the library fragments or from both ends (paired-end reads=PE). Longer reads are more expensive than shorter reads. Indexing (aka barcoding or tagging) is possible by using Illumina indexing adapters as well as custom adapters. The available read lengths are: PE35, SE75, PE75, SE150, PE150, PE250, and PE300.

Turnaround times vary and are dependent upon the running time of the sequencer and its availability.

GGBC is unique because we can help with services not widely available. Many scientists want to do things that aren’t perfectly amenable to standard protocols and we can help make it happen. We will determine what can be done at our end to achieve the goal and we will do it.

Important Resources

Explore Illumina Sequencing Methods

Illumina Sequencing Coverage Calculator

Obtaining FASTQ files off BaseSpace


Consultation and Assistance

Contact for Genomics and Bioinformatics Consultation

Please use this link to send an email to Dr. Magdy Alabady for consultation on new or existing Genomics and Bioinformatics projects. Also, you can contact Dr. Alabady for assistance with grant proposals and for obtaining a letter of support from GGBC.

For technical questions about existing or new Illumina projects, please contact the following Lab Managers/Staff;

  • Julia Portocarrero ( for read-to-sequence libraries, DNA-seq libraries, and Illumina instruments questions
  • Katarena Stoianova ( for microbiome and amplicon libraries
  • Casey Morrow ( for GBS libraries and for RNA-Seq and sRNA-Seq libraries

More contacts

Please visit our “All Inquiries” page for detailed information about who you should contact at GGBC to receive a quick and accurate response.

Sample Preparation

Libraries prepared with the following sample preparation kits at the GGBC can be used on any of Illumina’s next generation sequencing instruments including MiSeq, NextSeq 500 and HiSeq.

Crucial recommendations for all samples

  • Use fluorometric based methods for quantification (Qubit or PicoGreen) of the template DNA instead of UV spec based methods (e.g., NanoDrop) to obtain accurate DNA measurement.
  • DNA should have absorbance ratio values of 1.8–2.0.
  • RNA samples can be suspended in RNase-free water or 1X TE buffer prepared with RNase-free water.
  • RNA integrity should be assessed using the Agilent Bioanalyzer (or any similar system).
  • Please add any QC information (Qubit data, Agilent run, fragment analyzer run, gel picture,…) available to the online order form.
  • If you are are submitting 24 or more samples, the samples must be in a 96-well plate and include an Excel file with the sample layout in your order.

RNA is a sensitive material. RNA should be kept on dry or wet ice during transportation to the GGBC. Wet ice is recommended only for samples that are being transported locally within Athens. If you are traveling from farther away to submit your RNA samples, keep them on dry ice during transit. The GGBC will not accept RNA samples that are not stored properly during transit.

NGS stranded or unstranded RNA library preparation (Illumina compatible)  

In the stranded libraries the vast majority of the sequencing reads are from the first strand.  Selecting certain library types depends on the application. Generally speaking, all expression-by-sequencing analyses should use stranded libraries. Non-stranded RNA libraries can also be prepared upon request.  Feel free to contact us at to discuss your project.

Currently at GGBC, we use the Kapa Biosystems RNA library preparation chemistry for constructing of stranded libraries. We prepare both single- and dual-indexed libraries, depending on the target level of multiplexing. The quality of starting RNA is crucial for making good libraries and most importantly for robust data analysis. Starting material can be:

  •  100 ng – 4 μg of total RNA in < 50 µl
  •  10 – 400 ng of rRNA depleted or poly(A) – enriched RNA

RNA samples can be suspended in RNase-free water or 1X TE buffer prepared with RNase-free water. RNA integrity should be assessed using the Agilent Bioanalyzer (or any similar system). For sample’s concentration, we prefer using fluorometric methods, such as Qubit or Ribogreen.

NGS ribo-depleted stranded or unstranded RNA library preparation (bacteria)

Treatment of the total RNA from bacteria with the Ribo-Zero protocol removes cytoplasmic (nuclear-encoded) rRNA (5S, 16S, and 23S) prior to strand-specific or unstranded library preparation and sequencing. Please provide 1-5 µg of total RNA in RNase-free water.

TruSeq small RNA library preparation 

The TruSeq Small RNA sample preparation kit primarily targets microRNAs and other small RNAs, that have a 5’-phosphate and a 3’-hydroxyl group, to generate cDNA from total RNA or purified small RNA. Up to 48 samples can be multiplexed in one sequencing lane. Please provide 1 to 20 μg of high-quality total RNA prepared using a method that retains the small RNA fraction (Trizol etc.) at a concentration of at least 250 ng/µl in high quality water or 10 mM Tris buffer. Alternatively, submit the entire fraction of small RNA purified from 5-20 μg of total RNA in molecular grade water or 10 mM Tris buffer. Starting with enriched small RNA sample decreases the background signals, as it filters out most of the RNA degradation products.This kit supports only single-indexed libraries at the moment (up two 48 barcodes are available). RNA samples can be suspended in RNase-free water or 1X TE buffer prepared with RNase-free water. RNA integrity should be assessed using the Agilent Bioanalyzer (or any similar system). For sample’s concentration, we prefer using fluorometric methods, such as Qubit or Ribogreen.

NGS DNA library preparation (Illumina compatible) 

The KAPA Biosystems chemistry with in-house developed and validated adapters and indexing primers is used to prepare NGS DNA libraries for sequencing on any of the Illumina platforms. We refer to this system as “iTruS”. When multiplexing 24 or fewer libraries, we use the low throughput TruSeqLT to prepare 24 single-indexed libraries. For higher levels of multiplexing (more than 24 libraries), the high throughput (HT) iTruS is used to prepare dual-indexed libraries. The input DNA can be as low as 50 ng of high molecular weight DNA; however, we recommend submitting at least 300 ng of each sample in a maximum volume of 55 µl of TE.  The DNA integrity is very critical for producing a library that captures the complexity of the genome. The DNA integrity should be checked on a 0.7% TAE Agarose gel before submission. DNA should be submitted in 1XTE.

NGS DNA PCR-free library preparation (Illumina compatible) 

NGS DNA PCR-Free libraries eliminate PCR-induced biases. A minimum of 1 μg of input DNA in a maximum volume of 55 µl of TE is necessary to prepare a PCR-Free library.

Nextera XT DNA library preparation

The Nextera DNA sample preparation kit uses transposase to fragment the DNA and add adapters (known as tagmentation step) for single- or paired-end sequencing libraries. This kit is suitable for small genomes, such as prokaryotes, archae, and viruses. Also, it can be used to make libraries from PCR amplicons larger than 300 bp, plasmids, double-stranded cDNA, and concatenated amplicons. Up to 96 Nextera libraries can be multiplexed in the same sequencing lane/run. The main advantage of this kit is that it works with as low as 1 ng of starting DNA.

Amplicon (16S/ITS/custom) libraries

Please provide a minimum of 20 µl of high quality genomic DNA at a concentration of 5 ng/µl in nuclease-free water in a 96 well plate.  Also, provide the sequence of your target specific primers when submitting the order.

Ready-to-run libraries

Please provide ≥20 µl of your ready to run library at a 10 ± 4 nM concentration in 10 mM Tris pH 8. If you would like to use a custom primer for your MiSeq run, please submit ≥80 µl at a concentration of 10 mM. Please add any QC information (Qubit data, Agilent run, fragment analyzer run, gel picture,…) available to the online order form.

SMARTer Pico cDNA Synthesis

Synthesis of cDNA from 1-2 ng of total RNA. Minimum total RNA concentration for this cDNA synthesis kit is 20pg/uL in a 50uL reverse transcription reaction.

Fragment Analyzer QC: Electropherogram Reading and interpretation 

Fragment Analyzer QC: Electropherogram Reading and interpretation

Illumina Sequencing with Custom Primers

Custom sequencing primers can be used with non-standard Illumina sequencing assays. However, it is worth exploring if the assay can be converted to use standard Illumina sequencing primers, which are guaranteed to work.

Custom sequencing primers can be used with all reads: R1, R2, and I1 on the Miseq and NextSeq sequencers.

Custom primers designing consideration:

          • Must be positioned so that 5′–>3′ extension will occur using the sequence of interest as the template. The sequence of interest is either the fragment of interest or the index sequences
          • Must have properties which match those of Illumina’s primer as closely as possible:
            • Melting temperature (Tm): Calculate the Tm with the IDT Oligo Analyzer for the default buffer conditions (50 mM Na+). The minimum melting temperature depends on the platform, as following”
              • Miseq: Tm = 65°C
              • NextSeq: Tm = 60°C
            • Length = 33bp
            • GC content = 52%
          • Not have a risk of forming any significant 2° structures (i.e. won’t stick to itself, form loops, etc.)

Custom primers synthesis and submission considerations:

          • HPLC purified.
          • Concentration of100 uM.
          • Volume of at least 10ul.
          • Reconstituted in EB, DI water, etc. (we recommend a standard SSC buffer with TWEEN).
          • Submitted in a low-bind tube.

We require that primers be HPLC purified because HPLC removes incomplete sequences generated during oligo synthesis, while standard desalting does not.

Validating your custom sequencing primer

Before submitting your samples and custom primer, we recommend that you QC the primer. Sanger sequencing can be used to judge the primer’s annealing capabilities and rule out any adverse 2° structures. However, it is important to note that a primer and its corresponding sample may be successfully Sanger sequenced but still fail next generation sequencing. This is because of Illumina’s unidirectional method.

Therefore, we recommend setting up a PCR reaction using your custom sequencing primer and the P7 or P5 sequence. You will get product if both primers anneal effectively AND anneal to the correct end of the library fragment. By checking the product size versus the expected size, you can validate the primers.
PCR Primer combinations:
R1 + P7
R2 + P5
I1 + P5


Check the following documents:

Consideration when migrating non-Illumina Libraries between sequencing platforms

Illumina adapter sequencing guide

Prices and Quotes

Contact for Financial Inquiries and Quote Requests

Please email Kim and Elizabeth at, for financial inquiries or to request a quote. Be as specific as possible, so that they can more quickly assist you.

The information about the various components of the NGS workflow are separated in the following several tables including pricing for the various options.

Quick guide:
          1. Select the library type desired. See Table 1 for submission of individual samples for library preparation or Table 2 for large orders in which samples are submitted in a 96-well plate.
          2. Prepared libraries can be pooled by the GGBC staff (Table 3).
          3. Quality controls (concentration, fragment analysis, and qPCR) will be performed on the final library or library pool before loading the sequencer (Table 3).
          4. Depending on the amount of data needed, various run types on the MiSeq (Table 4) or NextSeq Mid- (Table 5) and High-outputs (Table 6) sequencers are available.
          5. A Quick Quote tool is available on the FBS Portal home page to easily get a cost estimate of your projects.
Illumina Library Preparation Costs

Table 1. Illumina library preparation costs (prices valid for libraries prepared AND sequenced at GGBC ONLY*)

Illumina Compatible
Library Type
Number of Libraries that can be multiplexed together based on kitUGA Fee
Non-UGA FeeNon-UGA FeeCommercial FeeCommercial Fee
(Price per library for up to 12)(Price for each additional library)(Price per library for up to 12)(Price for each additional library)(Price per library for up to 12)(Price for each additional library)
NGS Stranded or Unstranded RNA
(see Table 2)
24LT or 144HT$132$112$156$133$165$140
NGS Ribo-Depleted Stranded or Unstranded RNA Library (bacteria, yeast, human, rat, mouse)24LT or 144HT$195$195$231$231$244$244
NGS Small RNA48$115$92$136$109$144$115
(see Table 2)
24LT or 144HT$128$92$152$109$160$115
(see Table 2)
Nextera DNA Flex
(see Table 2)
NGS QuantSeq 3' mRNA FWD
(see Table 2)
cDNA from low input
RNA – SMRT technology
(Input 1-2 ng total RNA)

* Prices are significantly higher If libraries are to be prepared at GGBC but sequenced elsewhere. Please inquire for pricing.

Table 2. Illumina compatible library preparation reduced flat fees for samples submitted in a 96-well plate

llumina Compatible Library Type (submitted in 96 well plate)UGA FeeNon-UGA FeeCommercial Fee
NGS Stranded or Unstranded RNA$6,115$7,216$7,644
TruSeq Stranded Total RNA Gold (Human/Rat/Mouse) (up to 48 samples per plate)$8,59510,143$10,744
TruSeq Stranded Total RNA Gold (Human/Rat/Mouse) (49-96 samples per plate)$15,158$17,887$18,948
NGS DNA$7,639$9,015$9,549
NGS DNA-PCR free$7,639$9,015$9,549
Nextera DNA Flex$7,639$9,015$9,549
NGS QuantSeq 3’ mRNA FWD$2,541$2,999$3,177
Amplicons (16S/ITS/Custom)(up to 48 samples per plate)$607$717$759
Amplicons (16S/ITS/Custom)(49 to 96 samples per plate)$903$1066$1,129

Extra charges will be incurred if sample(s) do not pass QC and are resubmitted. Any extra sample(s) above 96 will be charged according to Table 1.


Library Pooling and Quality Control

Table 3. Library pooling and Quality control

Service DescriptionUGA FeeNon-UGA FeeCommercial Fee
Library Pooling up to 2-24 samples by qPCR$129$153$162
Library Pooling up to 25-48 samples by qPCR$179$212$224
Library Pooling up to 49-96 samples by qPCR$187$221$234
Library Pooling up to 97-144 samples by qPCR$230$272$288
Library Pooling up to 145-192 samples by qPCR$255$301$319
Library Pooling up to 193-288 samples by qPCR$281$332$352
Library Pooling up to 289-384 samples by qPCR$360$425$450
Pre-Sequencing QC (Qubit, FA, Kapa)$120$142$150
Additional Qubit quantification (per sample)$4.15$4.90$5.20
Additional Fragment Analyzer: High sensitivity NGS fragment analysis (per sample)$8.20$9.70$10.25
Additional Kapa qPCR (per sample)$49.00$57.85$61.25
MiSeq and NextSeq Flow Cells

Table 4. MiSeq flow cell

Run TypeMaximum number of reads passing filter
Maximum total number of
UGA FeeNon-UGA FeeCommercial Fee
MiSeq Nano (300 Cycles) (v2) flow cell;b SE75, PE75, SE100, PE100, SE150, and PE1502 300 Mb$319$377$399
MiSeq Nano (500 Cycles) (v2) flow cell; PE2502500 Mb$810$956$1,013
MiSeq Micro (300 Cycles) (v2) flow cell; PE1508 1.2 Gb$913$1,078$1,142
MiSeq (50 Cycles) (v2) flow cell; SE5012-15 750-850 Mb$1,361$1,606$1,702
MiSeq (150 Cycles) (v3) flow cell; SE150 22-25 3.3-3.8 Gb$1,465$1,729$1,832
MiSeq (150 Cycles) (v3) flow cell; PE7544-503.3-3.8 Gb$1,465$1,729$1,832
MiSeq (300 Cycles) (v2) flow cell; PE15024-304.5-5.1 Gb$1,637$1,932$2,047
MiSeq (500 Cycles) (v2) flow cell; PE25024-307.5-8.5 Gb$1,787$2,109$2,234
MiSeq (600 Cycles) (v3) flow cell; PE30044-50 13.2-15 Gb$2,264$2,672$2,830

Table 5. NextSeq Mid Output flow cell

Run TypeMaximum number of reads passing filter
Maximum total number of
UGA FeeNon-UGA FeeCommercial Fee
NextSeq (150 Cycles) SE150 Mid Output flow cellUp to 130 16.25-19.5 Gb$1,720b$2,030$2,150
NextSeq (150 Cycles) PE75 Mid Output flow cellUp to 26016.25-19.5 Gb$1,720b$2,030$2,150
NextSeq (300 Cycles) PE150 Mid Output flow cellUp to 260 32.5-39 Gb$2,503b$2,954$3,129

Table 6. NextSeq High Output flow cell

Run TypeMaximum number of reads passing filter
Maximum total number of
UGA FeeNon-UGA FeeCommercial Fee
NextSeq (75 Cycles) SE75 High Output flow cellUp to 400 25-30 Gb$2,198b$2,594$2,748
NextSeq (75 Cycles) PE35 High Output flow cellUp to 80025-30 Gb$2,198b$2,594$2,748
NextSeq (150 Cycles) SE150 High Output flow cellUp to 400 50-60 Gb$3,803b$4,488$4,754
Next Seq (150 Cycles) PE75 High Output flow cellUp to 80050-60 Gb$3,803b$4,488$4,754
NextSeq (300 Cycles) PE150 High Output flow cellUp to 800 100-120 Gb$5,826b$6,875$7,283

The GGBC performs MiSeq and NextSeq sequencing. Additional quality control assays might be requested before loading the instruments and these would incur some minor additional costs. Please inquire by contacting GGBC. Prices are subject to change without notice. To get a project cost estimate or to get a quote for a grant proposal, please contact GGBC.

a Consult Illumina for latest numbers.
b Additional 5% discount available for UGA customers doing 10 or more NextSeq runs between 7/01/19 and the end of the UGA fiscal year, 6/15/2020. Click here for more details.

Data Retrieval

Data distribution and retention policy at the Georgia Genomics Facility for Illumina sequencing:

          1.  At the end of your project, MiSeq and NextSeq sequencing data will be made available to you through Illumina BaseSpace (cloud storage) in FASTQ format. An invitation will be sent to you through e-mail, to transfer ownership of your run and project. Please accept this request within 30 days. Customers who utilize this service will have permanent access to their sequencing runs, and access to project results can be shared to collaborators at the owner’s discretion. You can register for an account and access this secure service at Basespace. The email used to share data through BaseSpace will be the address listed in the ‘Download the Data’ space on your order form. Please make sure that this email is the same used to set up your BaseSpace account.
          2. If your order was unable to be demultiplexed on BaseSpace, then we will demultiplex the data locally using the bcl2fastq pipeline. We use F*EX to transfer the data, and you will receive an email with a link to download the fastq files. If you would like to send the data to multiple emails, please specify this in the comments section of your order form.
          3. Alternatively, data can be downloaded onto a hard drive, which can either be provided by the customer or purchased through our facility. Additional shipping and handling charges may apply.
          4. Raw data is backed up and retained (offline) for a period of one year. If raw data must be extracted, additional fees will apply ($200 UGA, $236 Non-UGA, $300 Commercial).
          5. Data storage is also available through the Georgia Advanced Computing Resource Center (GACRC) for UGA researchers. Please contact them directly for more information.