A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries

Abstract

Genome targeting methods enable cost-effective capture of specific subsets of the genome for sequencing. We present here an automated, highly scalable method for carrying out the Solution Hybrid Selection capture approach that provides a dramatic increase in scale and throughput of sequence-ready libraries produced. Significant process improvements and a series of in-process quality control checkpoints are also added. These process improvements can also be used in a manual version of the protocol.

Figure 1

Overview of the hybrid selection method. Two specific sequencing targets and their respective capture baits are indicated in blue and red. (a) Generation of RNA bait capture probes. 150mer oligos are synthesized on array in batches of 55,000 and cleaved off. They are made double stranded by PCR amplification and tailed with a T7 RNA polymerase promoter, and RNA capture baits are made by transcription in the presence of biotinylated UTP. (b) Solution hybrid selection. RNA baits (from the top line) are mixed with a size selected pond library of fragments modified with sequencing adaptors. Hybridized fragments are then captured to streptavidin beads and eluted by the with-bead protocol for sequencing. See text for details.

Figure 2

With-bead SPRI method for pond library construction. SPRI magnetic beads are added to the sheared DNA sample. DNA is selectively bound to SPRI beads, which are immobilized when the sample plate is placed on a magnet, leaving other molecules in the liquid phase. The liquid phase is removed and discarded. The sample plate is then removed from the magnet and DNA is eluted from the beads. Library construction master mixes are then added to eluant/bead solution. The DNA and SPRI beads then pass through three cycles of reaction, binding to beads (in the presence of polyethylene glycol (PEG)/NaCl solution) and cleanup/washing. The cycles carry out end repair, A-base addition and adaptor ligation, respectively. A final elution is then followed by PCR amplification.

Figure 3

Yield output from pond library construction methods. Data are shown left to right, for pond libraries constructed with three methods: the widely used standard column-based cleanups [14], an automated implementation of standard bead cleanups and our implementation of with-bead SPRI cleanups. Each library was constructed with 3 μg input of NA12878 genomic DNA, in triplicate. Bars: total DNA output from pond library construction before PCR amplification. Blue diamonds: percentage recovery of input DNA for duplicates of 3 μg of the same input DNA. With-bead-based cleanups increased the amount of DNA retained throughout library construction compared to the standard column or SPRI cleanup methods.

Figure 4

Quality control checkpoints. (a-h) Eight different quality control checkpoints for the scaled SHS process are schematized. Quality is assayed at key steps to quickly identify failed samples and also to provide ability to troubleshoot process failures. See text for details. AFA, adaptive focused acoustics.

Figure 5

Increasing capacity over time and cumulative output. Bars show capacity for selections per week of protocols by date. Line shows cumulative hybrid selection captures performed.