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