Smrtbell template prep kit 1

The LR-PCR amplicons covering the entire CaPV genome were pooled in equimolar quantities to a final amount of 2 μg for single-molecule real-time (SMRT) sequencing (PacBio). SMRTbell libraries were prepared using the SMRTbell Template Prep Kit 1.0 (PacBio). To filter out smaller fragments and SMRTbell dimers, the libraries were size selected on a BluePippin 0.75 % Gel cassette (Sage Science) using the "High Pass V3″ protocol (collection protocol settings: 5.5 kb – 9.5 kb). P6-C4 sequencing was performed using a single SMRT cell on a PacBio RSII sequencer with a movie time of 240 min (PacBio) at the Genomics Core Leuven (KU Leuven, Belgium).

PacBio sequencing and analysis were conducted by OE Biotech Co. Ltd. (Shanghai, China). In brief, genomic DNA was extracted from bacteria using the CTAB method [22 (link)] according to the manufacturer’s instructions. The genomic DNA was subjected to quality control by agarose gel electrophoresis and quantified by Qubit (Invitrogen, Waltham, MA, USA). The library was constructed with the SMRTbell Template Prep Kit 1.0 (PacBio, Menlo Park, CA, USA). Single-molecule real-time (SMRT) sequencing was performed on the PacBio Sequel II platform using DNA/Polymerase Binding Kit 3.0 (New England Biolabs, Ipswich, MA, USA). Genome assembly was performed using SMRT Analysis 2.3.0 (Pacific Biosciences, Menlo Park, CA, USA).

To prepare long-chain genomic DNA isolated from JCM 15448, DNA extraction was performed as described previously [49 (link)]. Briefly, fungal cells were cultured in 10 ml RPMI1640 medium for 2 days, suspended in 450 μl Tris-EDTA (TE) buffer. The cell suspension was supplemented with 50 μl SDS and 500 μl phenol/chloroform, followed by bead-beating for 10 min by vortexing. After centrifugation at 17,400 x g for 5 min, the upper aqueous phase was subjected to electrophoresis on a 1% TAE agarose gel, followed by purification of long size DNA (15-40kb) using a Zymoclean large-fragment DNA recovery kit (Zymoresearch, Irvine, CA, USA). Short read and long read DNA libraries with purified DNA were constructed using QIAseq FX DNA Library Kit (QIAGEN, Hilden, Germany) and the SMRTbell template prep kit 1.0 (PacBio, Menlo Park, CA, USA) according to the manufacturer instructions, followed by whole-genome sequencing using NextSeq (Illumina, San Diego, CA, USA), MiSeq (Illumina), and Sequel (PacBio).

For A. tenebrosa, high molecular weight (HMW) DNA was extracted using two rounds of the method described for short-read DNA-seq from the same individual. HMW DNA was extracted from T. stephensoni according to the 10X Genomics salting-out protocol for DNA extraction from single insects [77 ]. In order to extract HMW DNA, minor modifications were made to this protocol, including the use of wide-bore pipette tips, low bind tubes and a gentle bead clean-up. DNA molecular weight was assessed using PippinPulse pulsed-field gel electrophoresis, while purity and quantity were determined using a Qubit fluorometer. Samples of sufficient purity (260:280 ratio of 1.8–1.9 and 260:230 ratio of 1.6–2.0) and quantity were sequenced.
Five micrograms of genomic DNA from each species was prepared using needle shearing and the BluePippin (SageScience) size-selection system. The SMRTBell® Template Prep Kit 1.0 (PacBio) was then used to prepare 14-kilobase libraries. Sequencing was undertaken on four sequencing chips using a 16-h run time on a PacBio Sequel®.

We performed full-length cDNA capture and isoform sequencing as previously described [3 (link)]. In brief, we designed a set of complementary oligonucleotide capture probes (Additional file 2: Table S8) to enrich for cDNA originating from NPIP paralogous copies and coupled this with a method to enrich for full-length cDNA molecules based on reverse transcriptase (RT) template switching [59 (link)]. Next, we generated PacBio Iso-Seq libraries and performed post-capture size selection to enrich for larger cDNA molecules according to the manufacturer’s guidelines (SMRTbell template prep kit 1.0, PacBio). SMRT sequencing was performed using the P6-C4 chemistry on the PacBio RS II instrument with 6-h movies [3 (link)]. A modified version of the Iso-Seq bioinformatics incorporating ToFU (Transcript isOforms: Full-length and Unassembled) was used for processing the long-read RNA-seq data (available at https://github.com/EichlerLab/isoseq_pipeline). Circular consensus sequence reads designated as putatively full length (if the expected terminal sequences and a poly(A) tract were observed) were then mapped to large-insert clone-assembled custom contigs using GMAP (v 2015-07-23) [60 (link)]. ORFs were identified using ANGEL (https://github.com/PacificBiosciences/ANGEL) and TRANSLATE as part of the ExPASy: SIB bioinformatics resource portal [61 (link)].

An aliquot of 250 μl of an overnight culture of the different EHEC O157:H7 strains was added to 20 mL of LB and incubated at 37°C with shaking for 3 ½ h. The bacteria were then harvested for DNA extraction using Genomic-Tip 100/g (Qiagen Inc Valencia, CA. DNA (10 ug/ml) were sheared to a targeted size of 30 kb using a g-TUBE (Corvaris, Woburn, MA) and subsequently concentrated using 0.45X volume of AMPure PB magnetic beads (Pacific Biosciences, Menlo Park, CA) according to the manufacturer’s protocol.
Sequencing libraries were created using 5 μg of sheared, concentrated DNA and the PacBio SMRTbell Template Prep Kit 1.0, according to the manufacturer’s protocol. Each library was sequenced using the RS II sequencing platform (Pacific Biosciences, PacBio) with the P6/C4 sequencing chemistry and the 360 min data collection protocol.