Illumina Sequencing

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

• GGBC (ggbc@uga.edu) for ready-to-sequence libraries, DNA-seq libraries, and Illumina instruments questions
• GGBC (ggbc@uga.edu) 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.

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.
• PLEASE USE V BOTTOM PLATES AND NOT ROUND/FLAT BOTTOM PLATES.

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	Description	Concentration requirement	Volume requirement
Stranded or unstranded RNA	We currently use Kapa Biosystems RNA chemistry for constructing stranded libraries. Non-stranded RNA libraries can be prepared upon request	4 - 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 RNA	Treatment 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 RNA	Primarily targets microRNA and other small RNA to generate cDNA from total RNA or purified small RNA	0.2 - 400 ng/uL of total RNA, or purified small RNA from 1-10 µg total RNA	≥ 10 uL
SMARTer Pico cDNA Synthesis	The 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
RRBS	Reduced 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
EM-Seq	NEBNext 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
GBS	Genotyping-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.

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

P5: 5′ AAT GAT ACG GCG ACC ACC GA 3′
P7: 5′ CAA GCA GAA GAC GGC ATA CGA 3′

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

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.