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, PE50, SE75, PE75, SE150, PE100, 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

Run Types and Expected Yield 


Consultation and Assistance

For technical questions about existing or new Illumina projects, please contact the following Genomics Professional;

  • Kateryna Stoianova ( for ready-to-sequence libraries, DNA-seq libraries, and Illumina instruments questions
  • GGBC ( for microbiome and amplicon 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, and NovaSeq. Note that a substantial variance in reads per library is to be expected when pooling genomic libraries obtained from diverse samples with varied genome sizes.

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 8 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.

If your samples do not meet the requirements in the table below, please contact us to discuss potential options for processing your project.

Illumina Library Prep

Library Prep Type
DescriptionConcentration requirementVolume requirement
Stranded or unstranded RNAWe currently use Kapa Biosystems RNA chemistry for constructing stranded libraries. Non-stranded RNA libraries can be prepared upon request4 - 160 ng/uL of total RNA, or 0.4 - 16 ng/uL of rRNA depleted or poly(A)-enriched RNA≥ 30 uL
Ribo-depleted stranded or unstranded RNATreatment of the total RNA to remove cytoplasmic rRNA prior to strand specific or unstranded library preparation. We currently use QIAseq FastSelect Fly, Yeast, Worm, Fish, Bacterial, Plant and HMR kits
>100 ng/uL of total RNA≥ 15 uL
Small RNAPrimarily targets microRNA and other small RNA to generate cDNA from total RNA or purified small RNA0.2 - 400 ng/uL of total RNA, or purified small RNA from 1-10 µg total RNA≥ 10 uL
SMARTer Pico cDNA SynthesisThe SMARTer Pico PCR cDNA Synthesis Kit provides a PCR-based method for producing high-quality cDNA from picogram quantities of total RNA.≥ 20 pg/uL of total RNA≥ 55 uL
NGS DNA (PCR)A fast, integrated workflow for a wide range of applications, from human whole-genome sequencing to amplicons, plasmids, and microbial species.30 pg/uL - 17 ng/uL of DNA≥ 35 uL
NGS DNA (PCR-free)A high-performing, fast, and integrated workflow for sensitive applications such as human whole-genome sequencing.1 - 12 ng/uL of DNA≥ 30 uL
seqWell purePlexDNA workflow for Low-pass whole genome sequencing, Whole small genome sequencing (<50 Mb), Metagenomics/Microbiome screening1 - 10 ng/uL of DNA≥ 10 uL
RRBSReduced Representation BisµLfite Sequencing allows for DNA methylation profiling at single-nucleotide resolution in CpG-rich regions of the genome.0.3 - 15 ng/uL of DNA≥ 40 uL
Whole genome bisulfite sequencing (WGBS)A streamlined workflow for making WGBS libraries. The procedure can accommodate ultra-low DNA input (including that derived from FFPE samples), making it ideal for methylation analysis of precious, limited, and target-enriched samples.0.5 pg/uL - 5 ng/uL of DNA≥ 25 uL
EM-SeqNEBNext Enzymatic Methyl-seq (EM-seq™) is a new method for identification of 5-mC and 5-hmC. Highly effective enzyme-based conversion minimizes damage to DNA and produces high quality libraries that enable superior detection of 5-mC and 5-hmC from fewer sequencing reads.0.2 - 4 ng/uL of DNA≥ 50 uL
GBSGenotyping-by-sequencing (GBS) uses restriction enzymes for targeted complexity reduction followed by mµLtiplex sequencing to produce high-quality polymorphism data at a relatively low per sample cost.> 200 ng of DNA≥ 40 uL
Ready-to-pool libraries/ Ready-to-run pool≥20 µl of your ready to run library at a 10 ± 4 nM concentration in 10 mM Tris pH 8. If you woµLd like to use a custom primers for your run, please submit ≥80 µl at a concentration of 10 uM.

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


NextSeq 2000 Custom Primers

Due to the construction of the NextSeq 1000/2000 cartridge, it is not possible to load custom primers into the Illumina primers. If Illumina primers are needed to sequence PhiX during a run, a NextSeq 1000/2000 Custom Primer kit is required. A charge of $704 will be added to the quote/invoice when using a custom read primer and a custom index primer for an Illumina NextSeq 2000 run.

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), and NextSeq 2000 (Table 5) 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.

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

Illumina Compatible
Library Type
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)
RNA libraries
NGS Stranded or Unstranded RNA
(see Table 2)
NGS Ribo-Depleted Stranded or Unstranded RNA Library $195$195$231$231$244$244
NGS Small RNA$115$92$136$109$144$115
NGS QuantSeq 3' mRNA FWD
(see Table 2)
cDNA synthesis
cDNA 2nd strand synthesis only (1 to 24 samples per plate)$447$528$559
cDNA 2nd strand synthesis only (25 to 48 samples per plate)$894$1,055$1,118
cDNA 2nd strand synthesis only (49 to 72 samples per plate)$1,341$1,583$1,677
cDNA 2nd strand synthesis only (73 to 96 samples per plate)$1,788$2,110$2,235
Double stranded cDNA synthesis (1 to 24 samples per plate)$598$706$748
Double stranded cDNA synthesis (25 to 48 samples per plate)$1,195$1,411$1,494
Double stranded cDNA synthesis (49 to 72 samples per plate)$1,793$2,116$2,242
Double stranded cDNA synthesis (73 to 96 samples per plate)$2,390$2,821$2,988
cDNA from low input
RNA – SMRT technology
(Input 1-2 ng total RNA)
DNA libraries
(see Table 2)
NGS DNA-PCR Free Library Prep (1-12 samples)$128$152$160
NGS DNA-PCR Free Library Prep (13 or more samples)$92$109$115
Enzymatic Methyl-Seq$89$89$106$106$112$112
Zymo-Seq RRBS$120$120$142$142$150$150

*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)$8,81010,396$11,013
TruSeq Stranded Total RNA Gold (Human/Rat/Mouse) (49-96 samples per plate)$15,537$18,334$19,422
QIAseq FastSelect Ribo-depletion 5S/16S/23S (96-well plate flat rate)$5,125$6,048$6,407
PurePlex DNA with UDI$3,137$3,702$3,992
NGS DNA-PCR free$7,639$9,015$9,549
NGS DNA **$7,830$9,240$9,788
Nextera Flex for Enrichment (up to 48 samples per plate)$7,019$8,283$8,774
Nextera Flex for Enrichment (49-96 samples per plate)$11,697$13,803$14,662
NGS QuantSeq 3’ mRNA FWD$2,541$2,999$3,177
Modified Amplicons (16S/ITS/Custom)(up to 48 samples per plate)$457$540$572
Modified Amplicons (16S/ITS/Custom)(49 to 96 samples per plate)$681$804$852

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.

**Illumina has changed the bead component in their library preparation kits and the Nextera DNA Flex products are now called Illumina DNA prep.


Table 3. Library pooling and Quality control

Service DescriptionUGA FeeNon-UGA FeeCommercial Fee
Library Pooling up to 2-24 samples$60$71$75
Library Pooling up to 25-48 samples$105$124$132
Library Pooling up to 49-96 samples$162$192$203
Library Pooling up to 97-144 samples$234$277$293
Library Pooling up to 145-192 samples$306$362$383
Library Pooling up to 193-288 samples$436$515$545
Library Pooling up to 289-384 samples$565$667$707
Pre-Sequencing QC (Qubit, FA, Kapa)$120$142$150
Additional Qubit quantification (per sample)$5.00$5.90$6.25
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


Table 4. MiSeq flow cells

Run TypeExpected single/paired-end reads passing filterExpected OutputUGA FeeNon-UGA FeeCommercial Fee
Nano Flow Cell
300 cycles1M / 2M300 Mb$836$937$1,045
Micro Flow Cell
300 cycles4M / 8M1.2 Gb$1,019$1,203$1,274
v2 Flow Cell
300 cycles12-15M / 24-30M4.5-5.1 Gb$1,771$2,090$2,214
500 cycles12-15M / 24-30M7.5-8.5 Gb$1,932$2,280$2,415
v3 Flow Cell
150 cycles22-25M / 44-50M3.3-3.8 Gb$1,598$1,886$1,998

Table 5. NextSeq 2000 flow cells

Run TypeExpected single/paired-end reads passing filterExpected OutputUGA FeeNon-UGA
P1 Flow Cell
100 cycles100M / 200M10 Gb$1,412.00$1,667.00$1,765.00
300 cycles100M / 200M30 Gb$1,717.00$2,027.00$2,147.00
600 cycles100M / 200M60 Gb$2,493.00$2,942.00$3,117.00
P2 Flow Cell
100 cycles400M / 800M40 Gb$1,858.00$2,193.00$2,323.00
200 cycles400M / 800M80 Gb$3,107.00$3,667.00$3,884.00
300 cycles400M / 800M120 Gb$3,977.00$4,693.00$4,972.00
600 cycles300M / 600M180 Gb$4,709.00$5,557.00$5,887.00
P3 Flow Cell
50 cycles1.2B / 2.4 B60 Gb$2,471.00$2,916.00$3,089.00
100 cycles1.2B / 2.4 B120 Gb$3,374.00$3,982.00$4,218.00
200 cycles1.2B / 2.4 B240 Gb$4,504.00$5,315.00$5,630.00
300 cycles1.2B / 2.4 B360 Gb$5,859.00$6,914.00$7,324.00

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. GGBC uses Globus to transfer the data. 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. Data storage is also available through the Georgia Advanced Computing Resource Center (GACRC) for UGA researchers. Please contact them directly for more information.