Titanium Paired End Sequencing allows the sequencing of DNA samples in paired ends separated by either approximately 3 kb, 8 kb or 20 kb. Paired End data is typically used to help order and orient contigs from a Shotgun sequencing project, this is also referred to as scaffolding. Therefore, Paired End sequencing is usually performed in parallel with the sequencing of a general library, prepared from the same DNA sample that was used to make the shotgun library but requires the preparation of a separate Paired End library. Such a Paired End library is composed of paired tags that are a known (approximate) distance from each other in the original sample. When the two tags of a pair can be mapped to two different assembly contigs, they specify the contigs order, orientation and approximate distance in the initial sample DNA.
Paired End Sample Requirements
The quality and quantity of the DNA sample are critical to the success of this procedure. Any contamination or degradation of the starting material will be directly reflected in the quality of the output library.
The procedure requires 5 μg of input DNA for making a 3 kb span, 15 μg of input DNA for making a 8 kb span and 30 μgof input DNA for making a 20 kb span Paired End library. Usage of lesser DNA amounts could result in poor quality or underperforming Paired End libraries. 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 preferably not be the result of any amplification (which may compromise representativity)
- DNA should not be degraded and should contain no particulate matter
- Input DNA size should be in pieces >15 kb for a 3 kb span, >24 kb for a 8 kb span and >60 kb for a 20 kb span Paired End library
- OD260/280 should be approximately 1.8
- Concentration should be at the minimum 200 ng/μl, in 10 mM Tris–HCL, pH 7.5–8.5, or Molecular Biology Grade water, not TE buffer, as higher concentrations of salt will alter the shearing characteristics of the sample
DNA quantitation using OD260 is not reliable. Input DNA concentrations must be verified by fluorometry. Other methods of quantitation often overestimate sample concentration, resulting in an inadequate amount of starting material. This could lead to poor library yield and reduced quality.