Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture

Abstract

Improvements in technology have reduced the cost of DNA sequencing to the point that the limiting factor for many experiments is the time and reagent cost of sample preparation. We present an approach in which 192 sequencing libraries can be produced in a single day of technician time at a cost of about $15 per sample. These libraries are effective not only for low-pass whole-genome sequencing, but also for simultaneously enriching them in pools of approximately 100 individually barcoded samples for a subset of the genome without substantial loss in efficiency of target capture. We illustrate the power and effectiveness of this approach on about 2000 samples from a prostate cancer study.

Figure 1.

Experimental workflow of the library preparation protocol for 95 samples for pooled hybrid capture.

Figure 2.

(A) Schematic overview of the library preparation procedure using the Illumina PE adapter (internal barcode in red). After a cascade of enzymatic reactions and cleanup steps, enrichment PCR can be performed to complete the adapter sites for Illumina PE sequencing (Rd1 SP, Rd2 SP are PE sequencing primers). Alternatively, libraries can be pooled for hybrid selection (if desired), and then enrichment PCR can be performed after hybrid selection. To achieve an even higher magnitude of pooling for sequencing, “indexing PCR” can be performed instead of “enrichment PCR,” whereby unique indices (in purple) are introduced to the adapter, and a custom index sequencing primer (index-PE-sequencing-Primer) is used to read out the index in a separate read. Finished libraries that have all the adapters necessary to allow sequencing are marked with an X. (B) Schematic figure of “daisy-chaining” during pooled solution hybrid capture, which may explain why a large proportion of molecules are empirically observed to be off-target when using long adapters. Library molecules exhibiting the target sequences hybridize to biotinylated baits, but unwanted library molecules can also hybridize to the universal adapter sites. The adapters of our “truncated” libraries (including barcode: 34 and 33 bp) are about half the length of regular “long” adapters (64 and 61 bases), and thus may be less prone to binding DNA fragments that do not belong to the target region.