Illumina Sequencing

Illumina sequencers deliver the most flexible and longest available reads of the shorter-read-length platforms, crossing important length thresholds to facilitate de novo sequence assembly. Steps to obtain sequences are as follow: 1) obtain/prepare appropriate template (DNA, RNA or small RNA), 2) prepare library by placing Illumina TruSeq adapters on the templates, 3) behind the scenes a technician will seed the DNA library onto a glass slide and sequence with the MiSeq, NextSeq, or HiSeq instrument, 4) image data are converted to sequences on the analysis server, and 5) data are delivered uploaded to an account on the basespace cloud or delivered to the customer via FTP, Hard Drive, etc.

Sequencing on an Illumina sequencer can be done by generating data from one Lillumina Logoillumina equipmentend (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.

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

GGF is unique because we can help with services not widely available. Many of our customers want to do things that aren’t perfectly amenable to standard protocols. We will help you to determine what can be done and help you to get it done.

 

Free Consultation

Contact for Financial Inquiries and Quote Requests

Please use this link belanger@uga.edu to send an email directly to Dr. Myriam Bélanger, Operations Director. In your email, include your full name, title, email address, institution, and a description of services that are needed. Be as specific as possible, so that she can more quickly assist you.

Contact for Genomics Consultation and NGS Technical Inquiries

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

Contact for Bioinformatics

GGF, as a part of the Quantitative Biology Consulting Group (QBCG), will provide bioinformatics support for your project. To request a free consultation, please contact Dr. Walt Lorenz, Lead Consultant, at 706-542-7974, or email at  qbcg@uga.edu, or fill out the Meeting Request Form.

Sample Preparation

Libraries prepared with the following sample preparation kits at the GGF 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.

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 ggf@uga.edu to discuss your project.

Currently at GGF, 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 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.

Prices

Table 1. Illumina library preparation costs

Illumina Compatible
Library Type
Number of Libraries that can be multiplexed together based on kit
UGA Fee

UGA Fee

Non-UGA Fee
Non-UGA Fee
Commercial Fee
Commercial 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$127$108$150$128$159$135
NGS Ribo-Depleted Stranded or Unstranded RNA Library (bacteria)24LT or 144HT$254$220$300$261$318$275
TruSeq Small RNA48$203$203$240$240$254$254
NGS DNA
(see Table 2)
24LT or 144HT$99$71$117$84$124$89
NGS DNA-PCR free
(see Table 2)
96$99$71$117$84$124$89
Nextera XT
(see Table 2)
96$99$71$117$84$124$89

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 of Unstranded RNA$5,880$6,939$7,350
NGS DNA$5,876 $6,934 $7,345
NGS DNA-PCR free$5,876 $6,934 $7,345
Nextera XT$5,876 $6,934 $7,345
Amplicons (16S/ITS/Custom)(up to 48 samples per plate)$416$491$520
Amplicons (16S/ITS/Custom)(49 to 96 samples per plate)$700$826$875
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 the above table.

Table 3. Prices for Library pooling and Quality control

Service DescriptionUGA FeeNon-UGA FeeCommercial Fee
Library Pooling up to 2-24 samples by qPCR$124.00$147.00$155.00
Library Pooling up to 25-48 samples by qPCR$172.00$203.00$215.00
Library Pooling up to 49-96 samples by qPCR$184.00$218.00$230.00
Library Pooling up to 97-144 samples by qPCR$225.00$266.00$282.00
Library Pooling up to 145-192 samples by qPCR$250.00$295.00$313.00
Library Pooling up to 193-288 samples by qPCR$275.00$325.00$344.00
Qubit quantification (per sample)$4.00$4.75$5.00
Fragment Analyzer: High sensitivity NGS fragment analysis (per sample)$7.90$9.35$9.90
Kapa qPCR (per sample)$47.00$55.50$58.75

Table 4. Prices for Illumina sequencing on MiSeq, NextSeq, or HiSeq instruments

Run TypeMaximum number of reads passing filter
(million)a
Maximum total number of
basesa
UGA FeeNon-UGA FeeCommercial Fee
MiSeq Nano (300 Cycles) (v2) flow cell; PE1502 300 Mb$908$1,072 $1,135
MiSeq Nano (500 Cycles) (v2) flow cell; PE2502500 Mb$1,026 $1,211 $1,283
MiSeq Micro (300 Cycles) (v2) flow cell; PE1508 1.2 Mb$1,192 $1,407 $1,490
MiSeq (50 Cycles) (v2) flow cell; SE5012-15 750-850 Mb$1,138 $1,343 $1,423
MiSeq (150 Cycles) (v3) flow cell; SE150 22-25 3.3-3.8 Gb$1,217 $1,437 $1,522
MiSeq (150 Cycles) (v3) flow cell; PE7544-503.3-3.8 Gb$1,217 $1,437 $1,522
MiSeq (300 Cycles) (v2) flow cell; PE15024-304.5-5.1 Gb$1,354 $1,598 $1,693
MiSeq (500 Cycles) (v2) flow cell; PE25024-307.5-8.5 Gb$1,472 $1,737 $1,840
MiSeq (600 Cycles) (v3) flow cell; PE30044-50 13.2-15 Gb$1,859 $2,194 $2,324
NextSeq (150 Cycles) SE150 Mid Output flow cellUp to 130 16.25-19.5 Gb$1,448b$1,709 $1,810
NextSeq (150 Cycles) PE75 Mid Output flow cellUp to 26016.25-19.5 Gb$1,448b$1,709 $1,810
NextSeq (300 Cycles) PE150 Mid Output flow cellUp to 260 32.5-39 Gb$2,095b$2,473 $2,619
NextSeq (75 Cycles) SE75 High Output flow cellUp to 400 25-30 Gb$1,813b$2,140 $2,267
NextSeq (75 Cycles) PE35 High Output flow cellUp to 80025-30 Gb$1,813b$2,140 $2,267
NextSeq (150 Cycles) SE150 High Output flow cellUp to 400 50-60 Gb$3,138b$3,703 $3,923
Next Seq (150 Cycles) PE75 High Output flow cellUp to 80050-60 Gb$3,138b$3,703 $3,923
NextSeq (300 Cycles) PE150 High Output flow cellUp to 800 100-120 Gb$4,796b$5,660 $5,995
HiSeq 2500 (v2) Rapidrun - SE50
(50 cycles; entire single flow cell: 2 non-
independent lanes)
Up to 300 12.5 - 15 GbPlease InquirePlease InquirePlease Inquire
HiSeq 2500 (v2) Rapidrun - SE100
(100 cycles; entire single flow cell: 2 non-independent lanes)
Up to 300 25-30 GbPlease InquirePlease InquirePlease Inquire
HiSeq 2500 (v2) Rapidrun - SE150
(150 cycles; entire single flow cell: 2 non-independent lanes)
Up to 300 37.5 - 45 GbPlease InquirePlease InquirePlease Inquire
HiSeq 2500 (v2) Rapidrun - PE50
(100 cycles; entire single flow cell: 2 non-independent lanes)
Up to 60025-30 GbPlease InquirePlease InquirePlease Inquire
HiSeq 2500 (v2) Rapidrun - PE100
(200 cycles; entire single flow cell: 2 non-independent lanes)
Up to 600 50-60 GbPlease InquirePlease InquirePlease Inquire
HiSeq 2500 (v2) Rapidrun - PE150
(300 cycles; entire single flow cell: 2 non-independent lanes)
Up to 600 75-90 GbPlease InquirePlease InquirePlease Inquire
HiSeq 2500 (v2) Rapidrun - PE250
(500 cycles; entire single flow cell: 2 non-independent lanes)
Up to 600 125-150 GbPlease InquirePlease InquirePlease Inquire

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

a Consult Illumina for latest numbers.
bAdditional 5% discount available for UGA customers doing 10 or more NextSeq runs between 7/01/16 and 6/09/17. 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 indefinite 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.
  2. 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.
  3. 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, $250 Commercial).
  4. Data storage is also available through the Georgia Advanced Computing Resource Center (GACRC) for UGA researchers. Please contact them directly for more information.
Bioinformatics

More information about related bioinformatics service.