Single molecule real time

Bioinformatic analyses were performed as described in the Supplementary Methods in S1 Text. The F11 genome was completely sequenced using a combination of long-read Pacific Biosciences Single Molecule Real-Time (SMRT) and short-read Illumina sequencing as previously described [42 (link),43 (link)]. Methods for DNA manipulation, rapid amplification of cDNA ends (5′ RACE), mutant construction, protein preparation, immunoblotting, whole-cell ELISA and β-galactosidase assays were performed as described in the Supplementary Methods in S1 Text.

PacBio Sequel Single Molecule Real-Time (SMRT) and Illumina sequencing platforms were used for genome sequencing. The complexity of the genome was assessed according to Illumina data. At least 5 μg of genomic DNA was required to construct a sequencing library for Illumina sequencing. DNA samples were cut into 400- to 500-bp fragments by using the Covaris M220 Focused Acoustic Shearer. The Illumina sequencing library was prepared from the cut fragments with the NEXTflex^TM Rapid DNA-Seq Kit. Simply put, the 5′ prime ends were the first end-repaired and phosphorylated. Next, the 3′ ends were A-tailed and connected to sequencing adapters. The third step is to use PCR technology to enrich adapter-ligated products. The paired-end Illumina sequencing (2 bp × 150 bp) was then performed on the Illumina HiSeq X-Ten machine using the prepared libraries.
For Pacific Biosciences sequencing, in a Covaris g-TUBE (Covaris, MA, United States), an Eppendorf 5424 centrifuge (Eppendorf, NY, United States) was used to rotate 8-μg DNA aliquots at 6,000 rpm for 60 s. SMRTbell sequencing adapters were used to purify, end-repair, and ligate the DNA fragments. The purification for the obtained sequencing library was repeated three times with Beckman Coulter genomics (MA) of 0.45 times volume as instructed by the manufacture. Next, a ∼10-kb insert library was prepared and sequenced on a SMRT cell.