Pacbio rs 2 system

Genomic DNA was extracted from ‐ 80°C frozen blood sample of a young male barn owl (M026801) using MagAttract HMW DNA Kit (Qiagen). In total five extractions quantified on a Qubit Fluorometer (ThermoFisher Scientific) yielded each between 1 and 6 µg of genomic DNA of a length between 35 and 50 kb (Fragment analyzer, Advanced Analytical, Labgene). In order to obtain a high‐quality de novo assembly, we combined libraries generated from standard short insert paired‐end libraries with that from mate‐pair libraries and PacBio long sequencing. Two mate‐pair libraries of 2 and 5 kb were prepared and sequenced by Fasteris (Fasteris), three TruSeq paired‐end libraries of 180 and twice 500 bp were prepared and sequenced at the Genomic Technologies Facility (GTF, University of Lausanne). The same high molecular weight DNA was used for PacBio libraries at the GTF. For PacBio, the DNA was sheared in a Covaris g‐TUBE (Covaris) to obtain 20 kb fragments. Then, the DNA size distribution was checked on the Fragment analyzer. 5 µg of the sheared DNA was used to prepare a SMRTbell library with the PacBio SMRTbell Template Prep Kit 1 (Pacific Biosciences) according to the manufacturer's recommendations. The library was sequenced on 37 SMRT cells with P4/C2 chemistry and MagBeads on a PacBio RSII system (Pacific Biosciences) at 240 min movie length.

Sequencing was performed using PacBio RS II (Menlo Park, CA, USA). Five microgram high molecular weight DNA without further fragmentation was used to prepare a SMRTbell library with PacBio SMRTbell Template Prep Kit 1 (Pacific Biosciences, Menlo Park, CA, USA) according to the manufacturer's recommendations. The resulting library was size-selected using a BluePippin system (Sage Science, Inc. Beverly, MA, USA) to enrich for molecules larger than 11 kbp. The recovered library was again damage repaired and then sequenced on a total of 25 SMRT cells with P6-C4v2 chemistry and by MagBead loading on the PacBio RSII system (Pacific Biosciences, Menlo Park, CA, USA) with 360 min movie length.

Total RNA (10 μg) was circularized with T4 RNA ligase 1⁸, digested with RNase R (Epicenter) to remove linear RNA, and termini were amplified with gene-specific primers listed in Supplementary Data 6. Two biological replicates of long-range single molecule real-time (SMRT) sequencing of 0.2–4 kb fragments was performed on a PacBio RS II system (Pacific Biosciences). Highly similar data sets were combined for final analysis. A single round of short-range sequencing was performed on a MiSeq instrument in 300 nt mode^{47 (link)}.

PacBio sequencing of total DNA of etiolated seedlings of O. elata was performed on a PacBio RS II sequencer (Pacific Biosciences, Menlo Park, CA, USA). For this, 5 μg of high molecular weight DNA (between 20 kb and 200 kb in size; see above) were used without further fragmentation to prepare five SMRTbell libraries with PacBio SMRTbell Template Prep Kit 1 (Pacific Biosciences, Menlo Park, CA, USA) according to the manufacturer’s recommendations. The libraries were additionally size-selected with BluePippin (Sage Science, Beverly, MA, USA) to enrich for molecules >10, 11 or 15 kb. Recovered libraries were again damage repaired and then sequenced on a total of 138 SMRT cells with P4-C2 or P6-C4v2 chemistry and by MagBead loading on the PacBio RSII system (Pacific Biosciences, Menlo Park, CA, USA) with 360 min movie length.

We prepared SMRTbell libraries for PacBio RS II sequencing following the manufacturer’s protocol for Multiplex SMRT Sequencing with PacBio Barcoded Adapters (Pacific Biosciences, Menlo Park, CA, USA). Prior to library preparation, amplicons of the three (A, B, C) or four (A, B, C, A-like) different loci, respectively, were pooled in equimolar amounts for each replicate of each individual. Each library contained ten such pooled samples with a DNA concentration of 100 ng per sample. Library preparations were performed according to supplier’s instructions except that after the damage repair step, we again added 1 μl of DNA Damage Repair Mix (Pacific Biosciences, Menlo Park, CA, USA) to each library and incubated the mixture for 30 min at 37 °C. This second damage repair step improved the sequencing by preventing early terminations in the sequencing process due to damages in the DNA template. Sequencing was performed on a PacBio RS II system (Pacific Biosciences, Menlo Park, CA, USA) using P6-C4 chemistry and 240 min movies.

To generate our own complete NZ reference genome, one isolate (F1157CHC) was further sequenced using the PacBio RSII system (Pacific Biosciences, CA, USA) as previously described¹⁹ . Gene prediction and annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (2013).