Table 1

Improvements to library construction process

Process step

DNA fragmentation

Size selection/clean-ups

Adapter ligation

Multiplexing

Library quantification

Standard method

Nebulization

Column-based; agarose gel cuts

Un-tagged or one of 12 multiplex identifiers (MIDs) in tubes

Up to 12 samples pooled after library construction process

Ribogreen ssDNA assay

Drawback

Low throughput; Reduced yield

Not easily automated; opportunity for sample mix-up

Low throughput

Limited pool complexity

Limited accuracy and sensitivity

Modified method

Acoustic shear in 96-well plate

Solid phase reversible immobilization in 96-well plates

120 barcoded adapters in plate format

Up to 120 samples pooled after adapter ligation or enrichment step

qPCR

Benefit

Improved yield;

increased throughput; automated setup

Amenable to automation; less opportunity for sample mix-up

Cross-contamination checks; high order multiplex within single region of PTP

Increased flexibility and pool complexity; decreased usage of LC reagents

Increased sensitivity; less input DNA required

LC, Library Construction; PTP, picotiter plate; qPCR, quantitative PCR; ssDNA, single-stranded DNA.

Lennon et al. Genome Biology 2010 11:R15   doi:10.1186/gb-2010-11-2-r15