Oxford Nanopore

Contact for Genomics and Bioinformatics Consultation

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

For technical questions about existing or new Nanopore projects, please contact:

• GGBC (ggbc@uga.edu)

For general questions, please review the rest of our website before contacting a lab member.

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.

General Submission Guidelines

• Please submit your order form before sending in samples.
• If you are submitting more than 24 samples, put the samples in a 96-well plate and include an Excel file with the sample layout in your order form.
• Clearly mark all sample submissions with your order number.
• Include any QC information (Qubit data, Bioanalyzer or Fragment Analyzer run, gel picture, etc) about your sample on your order form.
• DNA should have absorbance ratio values of 1.8 – 2.0, and be submitted in 1x TE.
• RNA samples should be suspended in RNase-free water, or 1x TE buffer prepared with RNAse-free water.

Preparation-Specific Guidelines

Libraries prepared at the GGBC can be sequenced on the Flongle or MinION Nanopore flow cells. If you have a project for Oxford Nanopore Sequencing and do not see a library preparation for your experiment, please contact us for custom sequencing.

We offer library preparation for gDNA, amplicons, cDNA, and direct RNA. Not all library preparation types are compatible with both flow cells. See the charts below for details about each type.

Sequencing can be done on either R9 version flow cells, or R10 version flow cells (see below for compatible libraries). Expected yield per flow cell is:

• Flongle (R9 and R10) – 1-2 Gb
• MinION (R9 and R10) – 15-30 Gb for DNA and 1-3 Gb for RNA

 

We offer library preparation for gDNA, amplicons, cDNA, and direct RNA. Not all library preparation types are compatible with both flow cells. See the charts below for details about each type.

R9 Flow Cell

Input	Library Prep Type	Kit	Input Requirement	Multiplexing
gDNA, amplicons, cDNA	Ligation	SQK-LSK109	1 µg of gDNA or 100-200 fmol of amplicons or cDNA	up to 96 samples (native ligation or PCR barcoding)
gDNA	Rapid	SQK-RAD004	400 ng	up to 96 samples
gDNA	Rapid PCR	SQK-RPB004	1-5 ng	up to 12 samples
gDNA, amplicons	PCR	SQK-PSK004	<100 ng gDNA	up to 12 samples
cells*, uHMW gDNA	Ultra-Long DNA (N50 > 50kb)	SQK-ULK001	6 million cells	no
polyA+ RNA, or total RNA	cDNA-PCR	SQK-PCS111	4 ng poly(A)+ RNA, or 200 ng total RNA	up to 24 samples
polyA+ RNA	direct cDNA	SQK-DCS109	100 ng poly(A)+ RNA	up to 96 samples
polyA+ RNA, or total RNA	direct RNA	SQK-RNA002	500 ng total RNA or 50 ng poly(A)+ RNA	no
gDNA	Targeted sequencing using CRISPR-Cas9	SQK-CS9109	1–10 µg gDNA (5 µg recommended)	no
gDNA	16S	SQK-RAB204	<10 ng gDNA per sample	up to 24 samples

*customers will have to perform DNA isolation following the recommended methods of the kit. GGBC will proceed with uHMW gDNA.

R10 Flow Cell Q20+

Input	Library Prep Type	Kit	Input Requirement	Multiplexing
gDNA, amplicons, cDNA	Ligation	SQK-LSK114	1 µg of gDNA or 100-200 fmol of amplicons or cDNA	up to 96 samples (native ligation or PCR barcoding)
gDNA	Rapid	SQK-RAD114	100 ng of gDNA	no
cells*, uHMW gDNA	Ultra-Long DNA (N50 > 50kb)	SQK-ULK114	6 million cells	no

*customers will have to perform DNA isolation following the recommended methods of the kit. GGBC will proceed with uHMW gDNA.

 

Core Methods

 

• Ligation
  • Offers a flexible method of preparing sequencing libraries from dsDNA (e.g. gDNA, cDNA or amplicons)
  • DNA ends are repaired and dA-tailed using the NEBNext End Repair/dA-tailing module before the sequencing adapters, supplied in the kit, are ligated onto the prepared ends
  • Recommended for users who want to optimize their sequencing experiment for accuracy and output, require control over read length, would like to utilize upstream processes such as size selection or whole genome amplification
• Rapid Ligation
  • Generates sequencing libraries from extracted gDNA in 10 minutes using a simple two-step protocol
  • At the heart of the kit is a transposase which simultaneously cleaves template molecules and attaches tags to the cleaved ends. Rapid Sequencing Adapters are then added to the tagged ends
  • Recommended for users who require a short preparation time, have limited access to laboratory equipment
• Ultra-Long DNA
  • Offers a means of preparing ultra-high molecular weight (uHMW) DNA for sequencing, which has shown to give N50s >50 kb and reads up to 4+ Mb
  • The kit is based on transposase chemistry: the transposase simultaneously cleaves template molecules and attaches tags to the cleaved ends. Rapid sequencing adapters are then added to the tagged ends. The last step is an overnight elution of the DNA library.
  • Recommended for users who are working with ultra-high molecular weight DNA and would like read lengths with an N50 >50 kb, require an end-to-end workflow to sequence ultra-high molecular weight DNA
• Rapid PCR
  • Offers the fastest and simplest method of preparation of barcoded libraries for low quantities of gDNA (1-5 ng), with only ~15 mins of hands-on preparation time
  • At the heart of the kit is a transposase which simultaneously cleaves template molecules in each sample and attaches tags, which contain primer binding sites, to the cleaved ends. The kit contains 12 primers which are then used to amplify each sample: each primer contains a barcode and 5’ tag which facilitates the ligase-free attachment of Rapid Sequencing Adapters. Amplified and barcoded samples are then pooled together and Rapid Sequencing Adapters are added to the pooled mix.
  • Recommended for users who wish to multiplex samples to reduce price per sample, have a low starting amount of DNA, require a simple library preparation procedure
• Direct RNA
  • Prepare any RNA with a 3’ polyA tail for 1D sequencing on the Oxford Nanopore sequencing devices
  • In contrast to cDNA sequencing protocols, which are used to prepare cDNA copies of RNA for sequencing, the Direct RNA kit is used to prepare RNA itself for sequencing. In Direct RNA sequencing, only the native RNA passes through the nanopore sensor thus the read length reflects the length of the RNA molecules in the sample.
  • Highly recommended for users who would like to remove RT or PCR bias, have transcripts that are difficult to reverse transcribe, are exploring attributes of native RNA such as modified bases
• Direct cDNA
  • Taking full-length messenger RNA, the complementary strand is synthesised using a kit-supplied oligo adapter. The RNA is then degraded, and the second (complementary) strand synthesised. Adapters supplied in the Direct cDNA Sequencing Kit are then ligated onto the cDNA, which introduce the components needed for the cDNA strands to enter the pore. One strand of the duplex is sequenced at a time, producing 1D reads.
  • Highly recommended for users who would like to identify and quantify full-length transcripts, are interested in differential gene expression, want to characterise and quantify isoforms, splice variants and fusion transcripts, wish to avoid bias and artefacts introduced through PCR
• Targeted sequencing using CRISPR-Cas9
  • Fast, flexible method for sequencing targets of interest without amplification
  • After DNA extraction, 5’ ends are dephosphorylated to reduce ligation of sequencing adapters to non-target strands. Cas9 ribonucleoprotein particles (RNPs), with bound crRNA and tracrRNA, are added to the genomic DNA, and bind then cleave the region of interest (ROI). dsDNA cleavage by Cas9 reveals blunt ends with ligatable 5’ phosphates. All of the DNA in the sample is dA-tailed, which prepares the blunt ends for sequencing adapter ligation. Sequencing adapters are ligated primarily to Cas9 cut sides, which are both 3’ dA-tailed and 5’ phosphorylated. The library preparation is cleaned up to remove excess adapters using AMPure XP beads and resuspended in Sequencing Buffer (Non-target molecules are not removed). The subsequent library preparation is added to the flow cell for sequencing.
  • Recommended for users who want to target specific regions of 5-20 kb to high coverage (>100x) on a single MinION Mk 1B flow cell without PCR; want to sequence a larger, contiguous Region of Interest (ROI), using up to 100 target sites, in a single assay; have 1-10 µg of available high quality gDNA; are interested in methylation patterns or other modified bases; have gene targets that are highly repetitive, or wish to evaluate the number of repeats in an expansion, where traditional amplification method or sequencing-by-synthesis methods could yield a biased result; want to sequence long gene targets in a single pass that are not amenable to long-range PCR (>30 kb)

Table 1. Oxford Nanopore single sample library preparation fees

Library Type	UGA Fee	Non-UGA Fee	Commercial Fee
Direct RNA single sample library prep	$455	$537	$569
PCR free cDNA seq single sample library prep	$302	$357	$378
cDNA seq single sample library prep. PCR included.	Coming soon	Coming soon	Coming soon
DNA single sample library prep with rapid ligation	$215	$254	$269
DNA single sample library prep with ligation	$290	$343	$363
Amplicon single sample library prep with ligation starting from amplicon	$302	$357	$378
Amplicon single sample library prep with ligation starting from gDNA	$598	$706	$748

Table 2. Oxford Nanopore multiplexed samples library preparation fees

Library Type	UGA Fee	Non-UGA Fee	Commercial Fee
Multiplexed DNA library prep with ligation (per sample)	$116	$137	$145
Multiplexed DNA library prep with ligation (per pool)	$159	$188	$199

Table 3. Oxford Nanopore multiplexed amplicons library preparation fees

Library Type	UGA Fee	Non-UGA Fee	Commercial Fee
Multiplexed 2-48 amplicons library prep starting from gDNA	$1,102	$1,301	$1,378
Multiplexed 49-96 amplicons library prep starting from gDNA	$1,729	$2,041	$2,162
Multiplexed 2-48 amplicons library prep starting from amplicon DNA	$697	$823	$872
Multiplexed 49-96 amplicons library prep starting from amplicon DNA	$1,088	$1,284	$1,360

Table 4. Oxford Nanopore sequencing run pricing

Run Type	UGA Fee	Non-UGA Fee	Commercial Fee
Flongle flow cell	$205	$242	$257
1D flow cell	$1,083	$1,278	$1,354

1. GGBC uses Globus  to transfer the data. This will include the fast5 and fastq files from reads that passed and failed quality checks, duty time and throughput reports, and Nanopore run summary files.
2. Basecalling is done with a fast basecalling algorithm by default. Normally, you would want to rerun it for a more accurate result with a high-accuracy basecalling. This can be done using fast5 files that we provide you with. An additional $300 charge will apply if you need GGBC to rerun basecalling for you instead.
3. The data files will be permanently deleted from GGBC’s data storage after 6 months from the data transfer date.