M220 focused acoustic shearer

Genomic DNA was isolated from the overnight liquid cell suspension of E. roggenkampii strain by Wizard Genomic DNA Kit (Promega). DNA quality and concentration were estimated by TBS-380 fluorometer (Turner BioSystems Inc., Sunnyvale, CA, United States) and DNA with high quality (OD₂₆₀/₂₈₀ = 1.8 ∼ 2.0 > 20 μg) was employed for additional experiment. The genome was sequenced by a fusion of Nanopore and Illumina sequencing platforms. The Illumina data were employed to assess the complexity of the genome. For Illumina sequencing, as a minimum 1 μg genomic DNA was utilized for every isolate in the assembly of the sequencing library. DNA fragments were incised into 400–500 bp by a Covaris M220 Focused Acoustic Shearer. Illumina sequencing libraries were prepared by NEXTflex Rapid DNA-Seq Kit. Briefly, 5′ prime ends were first end-repaired and phosphorylated. Next, the 3′ ends were A- tailed and ligated to sequencing adapters. The third step was to enrich adapters-ligated products using PCR. The organized libraries were used for paired-end Illumina sequencing (2 × 150 bp) on an Illumina HiSeq X Ten. For Nanopore sequencing, 15 μg of genomic DNA was spin in a Covaris G-TUBE (Covaris, MA) to cut the genomic DNA into ∼10 kb fragments, then performed magnetic bead purification and connect the sequencing adapters to both ends.

The genome was sequenced using a combination of PacBio Sequel Single Molecule Real Time (SMRT) (Pacific BioSciences, Menlo Park, MA, USA) and Illumina (Illumina, San Diego, CA, USA) sequencing platforms (Illumina, San Diego, CA, USA). For Illumina sequencing, at least 5 μg genomic DNA was utilized for sequencing library construction for each strain. The DNA samples were fragmented into 400–500 bp fragments using a Covaris M220 Focused Acoustic Shearer (Covaris, Woburn, MA, USA). The sheared fragments were subsequently utilized to generate Illumina sequencing libraries using the NEXTflex™ Rapid DNA-Seq Kit (Bioo Scientific, Austin, TX, USA). The 5′ ends of the fragments were first end-repaired and phosphorylated, while the 3′ ends were A-tailed and ligated to sequencing adapters. The adapter-ligated products were subsequently enriched through PCR. Subsequently, the prepared libraries underwent paired-end Illumina sequencing (2 × 150 bp) using the Illumina HiSeq X Ten machine (Illumina, San Diego, CA, USA).
The aliquot of 8 μg DNA was spun at 6000 rpm/min for 60 s. The DNA fragments were purified and end-repaired. The resulting sequencing library was purified three times using 0.45 times the volume of Agencourt AMPure XP beads (Beckman Coulter Genomics, Brea, MA, USA). Finally, an ~10 kb insert library was prepared and sequenced.

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.