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Genomic DNA Sequencing

Whole genome sequencing allows sequencing and structural variation detection of viruses, bacteria and other microbes, fungi, animals and plants, whole genome amplified or randomly amplified DNA, purified DNA such as BACs, fosmids and plasmids.

Shotgun Sample Requirements

The procedure requires 500 ng of sample DNA but we request at least 1 μg in case of problems. Ideally the DNA should be checked to ensure it is derived from the target organism and contains no other contaminating DNA.
At a minimum, the DNA sample should meet the following criteria:

  • DNA must be double stranded
  • DNA should not be from an amplified library (which may compromise representativity)
  • DNA should not be degraded and should contain no particulate matter
  • Input DNA size should be in pieces >1.5kb
  • OD260/280 should be approximately 1.8
  • Concentration should be 50 ng/μl or above, in TE

Because DNA quantitation using OD260is variable and dependent upon DNA purity, the sample DNA concentration should be checked by fluorometry or gel electrophoresis on a 1–2% agarose gel using a DNA Mass Ladder.

A variation on the standard procedure allows the preparation of ssDNA libraries from Low Molecular Weight DNA samples. LMW DNA is a sample comprised of fragments in the 70–500 bp range, such as short sequence tags, PCR products, cDNA derived from microRNAs, etc.