Guidelines for submission of genomic DNA to the DOE Joint Genome
Institute

The JGI has sequenced over 650 prokaryotic, eukaryotic and metagenomic
projects and finished over 250 genomes. DNA quality (molecular weight
and purity) and quantity have always been the two most critical factors
in the success of the sequencing projects. If you cannot meet the JGI
specifications for DNA or have questions or concerns, please contact
your project manager or <jgi_project_management@lanl.gov>

**[JGI Starting Material Specifications: Quality: (1)
purity]{.underline}**

For prokaryotes, the starting culture should be axenic, strain pure, and
started from a single colony or from a culture diluted to extinction. It
may not be possible to assemble a genome that is not derived from an
axenic and strain pure culture. If an axenic and strain pure culture is
not possible, description and justification is required.

For eukaryotes, the starting DNA should be free of contaminants and/or
symbionts, should have \< 5% organelle DNA, and \< 1% polymorphism
wherever possible. If a haploid form for the organism exists this is
preferable. It may not be possible to assemble a genome that is derived
from a contaminated or polymorphic sample.

Watch out for other types of contamination -- RNA blobs and discrete
bands require clean-up. Please refer to the recommended RNAse I clean-up
protocol: <http://my.jgi.doe.gov/index.html>. In some cases, the
presence of some impurities (polysaccharides or proteins) can be
predicted by examining the way DNA fragments migrate (or do not migrate)
in an agarose gel. If most of the DNA fragments form a streaky pattern
or get stuck on top of the wells, the level of impurities contamination
may cause problems in the shearing and the clonability of the DNA.

**[JGI Starting Material Specifications: Quality: (2) molecular
weight]{.underline}**

Molecular weight of the DNA sample decides the insert size of the
library we can construct. Most of the DNA preparation protocols should
be able to generate around 100Kb fragment size DNA. For projects in need
of a fosmid library we request that the majority of the DNA fragments be
greater than 40Kb in size. JGI will run a pulsed-field gel to determine
the sample size range.

For other types of libraries (small insert size Sanger, 454 Titanium
paired-end, etc), we believe that HMW DNA will give a better chance of
constructing a high quality library. We will not generally accept
samples with most DNA fragments smaller than 20Kb in size or heavily
degraded DNA samples; please contact your project manager to discuss
this requirement.

**[JGI Starting Material Specifications: Quantity]{.underline}**

For Sanger sequencing, we request 5 ug of DNA for constructing a 3Kb
library, 10 ug of DNA for constructing an 8Kb library, and 30 ug of DNA
for a fosmid library. Additional sample is needed if more than one
attempt of library construction is needed. For a eukaryotic whole genome
shotgun project, we generally request 100 ug of DNA, enough for at least
two attempts of each of the three insert size libraries. For plant
genomes, we request even more DNA because multiple fosmid libraries are
usually needed.

For 454 sequencing, 5 ug is needed for each Titanium shotgun library and
15 ug is needed for each Titanium paired-end library.

For Illumina sequencing, 5 ug is needed for each library.

The current process for sequencing prokaryotic genomes includes the
construction of 3 libraries (Titanium shotgun, Titanium paired-end, and
Illumina). We generally request 50 ug of prokaryote DNA so that we would
have enough DNA for two attempts of each of the three libraries. The
concentration of the DNA must be \>= 50 ng/ul as judged by agarose gel /
mass standards.

Please discuss with your project manager if you do not have enough
material. We may be able to work with the DNA you have as long as
efforts are ongoing to prepare more DNA. We realize these DNA quantities
may not be possible for environmental samples, but the project will have
the best chance for success if this amount can be sent. In some cases,
JGI may be able to amplify the DNA.

**[JGI Starting Material Specifications: Shipping]{.underline}**

Prior to shipping samples to the JGI, the Collaborator Sample
Information (CSI) form must be completed and submitted on-line
([https://my.jgi.doe.gov/csi/user/login]{.underline}). A well-labeled
gel image of your DNA prep is required as part of the submission. See
the "Genomic_DNA_QC_Guides.pdf" file for detailed instructions on
running the QC gel, or find the complete JGI protocol at
[http://my.jgi.doe.gov/index.html](http://www.jgi.doe.gov/sequencing/protocols/index.html)*.
Gel images that do not contain the appropriate size and mass standards
will not be approved for shipping.*

Format for shipping in order of preference:

1.  Resuspended in TE (10 mM Tris, ph 7.5 -- 8, EDTA 0.1 mM) at \>= 50
    ng/ul shipped on dry ice

2.  Ethanol pellet shipped at RT or on wet ice. Note some ethanol
    pellets the JGI has received will not spin down and resuspend.

3.  Dry pellet shipped at RT or on wet ice. Note that most dry pellets
    the JGI has received will not resuspend.

*DNA should be shipped to the JGI in **one tube**.* The tube should be
legibly labeled with genus, species, strain, date and project number
(this will be provided after the sample has been approved for shipping).
If the DNA is prepared in multiple preps, each prep should be QC'ed
separately, and good preps pooled into one tube for shipping to the JGI.

**Please Note:** JGI procedures such as CTAB for isolating prokaryotic
HMW gDNA and various eukaryotic HMW protocols are available on the
following website:
[http://my.jgi.doe.gov/index.html](http://www.jgi.doe.gov/sequencing/protocols/index.html)