Truseq nano dna library prep kit for neoprep

Nucleic acids were extracted from the isolates of members of the genus Streptoccocus using a DNeasy Blood and Tissue Kit (QIAgen) or a MaxMAX DNA Multi-Sample Ultra Kit (ThermoFisher). The extracted DNA was made into Illumina-compatible libraries using either a Nextera XT (Illumina), TruSeq Nano DNA Library Prep Kit for NeoPrep (Illumina) or a NxSeq AmpFREE Low DNA Library Kit (Lucigen). Libraries made with the NxSeq AmpFree DNA Library Kit were quantified using the NEBNext Library Quant Kit for Illumina (New England BioLabs). All libraries were sequenced on an Illumina MiSeq using a 500-cycle MiSeq V2 kit (Illumina). Quality of the raw sequencing reads was assessed using FastQC v0.11.5 (www.bioinformatics.babraham.ac.uk/projects/fastqc/) and MultiQC 1.2 [12 (link)]. One isolate, BCCDCPHL-Ssp027, failed to sequence and was not further analyzed. All raw sequence data is available from the BCCDC PHL Genomic Data Bank (BioProject: PRJNA379148), specifically this study under BioProject: PRJNA428833.

Total DNA of Pseudokirchneriella subcapitata (NIES-35) was extracted using DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following manufacture’s protocol. The DNA was fragmented to approximately 550-bp segments using a Covaris M220 ultrasonicator (Covaris, Woburn, MA, USA). The genomic library was constructed using a TruSeq Nano DNA library prep kit for NeoPrep (Illumina, San Diego, CA, USA) and sequenced by the MiSeq platform (Illumina) using the 600-cycle MiSeq reagent kit version 3. Low-quality reads/bases were filtered using Trimmomatic version 0.36 [18 (link)]. De novo assembly was performed using SPAdes 3.9.0 [19 (link)]. A sequence of plastid-encoded Rubisco large subunit (rbcL) was obtained and deposited on DNA Data Bank of Japan (DDBJ) under the accession number LC200423. Basic Local Alignment Search Tool (Blast) [20 (link)] was used to verify the identity of the strain NIES 35. The sequences of rbcL were aligned using MUSCLE [21 ] integrated into MEGA7 [22 ]. Nucleotide substitution model selection and construction of a maximum likelihood tree were carried out by using MEGA7 [22 ]. GTR+G model was chosen in this analysis. Bootstrap values were calculated with 1,000 pseudoreplicates.

For DNA libraries, we prepared paired-end (~550 bp insert) and mate-pair (~3–4 kbp insert) libraries. The paired-end library was prepared using the TruSeq Nano DNA Library Prep Kit for NeoPrep (Illumina, San Diego, CA, USA) with the NeoPrep system (Illumina) following the manufacturer’s protocol. The mate-pair library was prepared using the Nextera Mate Pair Sample Preparation Kit (Illumina) following the manufacturer’s protocol. We also prepared a paired-end RNA library (~550 bp insert) using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England BioLabs, Inc., Ipswich, MA, USA). mRNA was purified using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs). All libraries were sequenced on the MiSeq sequencing system (Illumina) using the MiSeq Reagent Kit v3, 600 cycles (300 bp × 2), and the MiSeq Reagent Kit v2, 150 cycles (75 bp × 2), for the paired-end and mate-pair libraries, respectively.