University of Idaho - IBEST
Genomics Resources Core
INSTRUCTIONS for 454 Sequencing Sample Submission
Aug 2012 - Version 1.0
The University of Idaho – IBEST Genomics Resources Core wishes to work with
investigators to ensure they receive good data and the correct data to answer the
biological questions intended for the project. As such, core personnel request that
investigators:
1. Consult a core bioinformatician as to the projects goals, design, and replication
as early as possible in the project (preferably before sample collection).
2. Consult a core molecular specialist during sample preparation in order to
ensure optimal quality data.
Contact:
Bioinformatics: Matt Settles 1-208-885-6051
Laboratory: Suresh Iyer and Dan New 1-208-885-7023
Submitting Samples to the IBEST Genomics Resources Core
Documentation


ALL samples need to be accompanied by a completely labeled gel (e.g. 1, 2, 3) and ladder,
with the sizes of each band of the ladder marked clearly.
◦ Gels should be high quality regarding camera resolution, band intensity, band separation,
ladder separation, and sufficient quantity of nucleic acid.
◦ Laser printer copies do not provide the resolution needed.
◦ Each sample should be clearly labeled both on the gel image and on the tube. In addition to
labeling your tubes with your sample ID, please also include a numerical tube ID 1-99 on
the tube.
A completed "454 Sequencing sample submission sheet" (provided) is also required, both
email and physical copy accompanying the samples.
Shipping

Please ship samples overnight by FedEx on dry-ice to
c/o Daniel New
c/o Suresh Iyer
University of Idaho
IBEST Genomics Resources Core
Life Science South Rm 252
Moscow, ID 83844-3051

All samples to be accompanied by a complete “454 Sequencing sample submission sheet”.
Below is a guide to quantity and quality of sample, or if you have an uncommon sample type
please call the core laboratory.
High quality DNA is required for successful library generation and high quality Illumina sequencing.
DNA can be extracted using the DNA extraction method of your choice. Samples should be delivered
preferably in Tris-HCl 10mM, pH 8.5 with 0.1% Tween 20 (or Qiagen EB), or in 1X TE buffer (10 mM
Tris-HCl and 0.1 mM EDTA, pH 8.0), free of any particulate matter, and in 1.5mL tubes.
DNA – Submission
Sample type
Quantity
Double Stranded DNA -
1 μg in ≤ 100l
buffer

As quantified by Fluorometer. If quantifying using a nanodrop/spectrophotometer,
then at least 2 μg (in a maximum of 100l TE buffer) is required.
Spectrographic QC of DNA (Nanodrop) (Highly Recommended)



DNA concentration ≥ 5ng/µL (A260 x 50 x Dilution Factor)
A260/A280 ≥ 1.8
A260/A230 ≥ 1.8
Amplicon Sequencing
High quality PCR product is required for high quality sequencing. PCR products should only be
generated in consultation with the Core, size of the PCR product should not exceed 800bp.
Samples should be delivered in 1X TE buffer (10 mM Tris-HCl and 0.1 mM EDTA, pH 8.0), and free of
any particulate matter. The amplicons generated should preferably contain no secondary bands or
primer dimers visible. Please submit samples in 1.5ml tubes only.
DNA – Submission
Sample type
Quantity
Double Stranded PCR product -
100ng of each PCR
product

As quantified by fluorometer (consult the core if you do not have access to a
fluorometer)
Agarose Gel and/or Bioanalyzer QC of DNA

Verify PCR samples are of desired molecular weight

Samples must not be degraded

No secondary bands or primer dimers should be visible.

PCR reactions should be done in 50ul volume preferably.

10% of PCR product should be loaded and run on a 2% agarose gel.

Agarose gels should be run without Ethidium bromide (EtBr) in 0.5xTAE buffer also without
EtBr. Place gels in a 1ug/ml EtBr bath for staining (~30 min). Remove and then briefly destain with
water (~5 min). Stained gels are then viewed and photographed.
III. Transcriptome Sequencing
High quality RNA is required for optimal cDNA synthesis yield and Roche 454 library generation. RNA
can be extracted using the RNA extraction method of your choice and should be followed by
DNAse treatment prior to submission (we recommend the Ambion Turbo-DNA-free kit).
RNA – Submission
Sample type
Quantity
RNA
10µg total RNA of
each sample
OR
≥ 200ng mRNA of
each sample



The following values are post DNAse Treatment and as quantified by Nanodrop.
Sufficient amount of total RNA is needed to get 200ng mRNA post mRNA isolation.
Eukaryotic mRNA is ~ 1-5% of total RNA. Please keep this in mind if submitting total
RNA.
Prefer 1µg of mRNA when possible.
Spectrographic QC of RNA (Nanodrop) (Highly Recommended)

RNA concentration ≥ 11 ng/µL (A260 x 40 x Dilution Factor) in 19 µL Molecular Biology Grade Water


A260/A280 ≥ 1.8
A260/A230 ≥ 1.8
Bioanalyzer and/or Agarose Gel QC of RNA


Verify RNA samples are of sufficient molecular weight
Samples are not degraded, degraded samples will not generate good quality cDNA for 454
sequencing.
Bioanalyzer: Recommended
 RIN (RNA Integrity Numbers via Bioanalyzer) ≥ 6.0
 28s/18s ≥ 1.5