Complete viral RNA genome sequencing of ultra-low copy samples by sequence-independent amplification

Malboeuf, Christine M.; Yang, Xiao; Charlebois, Patrick; Qu, James; Berlin, Aaron M.; Casali, Monica; Pesko, Kendra N.; Boutwell, Christian L.; DeVincenzo, John P.; Ebel, Gregory D.; Allen, Todd M.; Zody, Michael C.; Henn, Matthew R.; Levin, Joshua Z.

We prepared paired-end libraries for Illumina sequencing according to previously published methods (35) . For Ovation RNA-Seq version 1 reactions, no shearing or size selection was performed since the majority of the amplified products were between 200 and 600 bp. For Ovation RNA-Seq version 2 reactions, amplified products were sheared in a single tube format using an adaptive focused acoustic shear technology (S2 machine, Covaris, Woburn, MA). Shearing conditions were as follows: time = 180 seconds, duty cycle = 10, intensity = 5; cycles per burst = 100, mode = frequency sweeping. Sheared samples were purified using 2.2 volumes of AMPure XP beads resulting in fragments between 150 and 500 bp. Briefly, libraries were prepared by end-repair of the products of the NuGEN reactions (sheared for version 2) followed by A-base addition, adapter ligation, and PCR. The following modifications were made. The libraries were generated with forked adapters containing unique 8-base index sequences. The number of PCR cycles varied from 8 to 15 cycles per sample, using the lowest number of cycles needed for sequencing. The PCR reactions were purified using 2 rounds of 0.7 volumes of AMPure XP beads. We sequenced the HIV and WNV indexed libraries in pools of 12 to 36 samples on a HiSeq 2000 (Illumina, Hayward, CA; 1 lane; 101 base paired-end reads). We sequenced the RSV indexed libraries in a pool of two samples on a MiSeq (Illumina; 101 base paired-end reads). Sequence reads were binned by index read prior to further analysis.