Table 1 Improvements to library construction process
From: A scalable, fully automated process for construction of sequence-ready barcoded libraries for 454
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 |