Neb blunt ta ligase

Genomic DNA (1.5 μg) was end-repaired using the NEBnext Ultra II End Repair kit (New England Biolabs, Ipswich, MA, USA) and cleaned up with 1x volume of AMPure beads (Beckmann Coulter, USA). Adapter ligations were performed for 20 minutes using NEB blunt/TA ligase (New England Biolabs). The library mixtures were cleaned up using 0.4X AMPure beads (Beckmann Coulter) and eluted in 25 μl of elution buffer. The eluted library was used for sequencing. Whole-genome libraries were prepared using the ligation sequencing SQK-LSK108 Oxford Nanopore sequencing kit (ONT). Sequencing was performed on a MinION Mk1b (ONT) using SpotON flow cell (FLO-MIN106) in a 48-hour sequencing protocol on MinKNOW (version 1.1.20, ONT).

The extracted DNA was purified using Qiagen DNeasy Blood & Tissue kit column (Qiagen, Germany). Paired-end (PE) and mate-pair (MP) sequencing libraries with insert sizes of 400–550 bp and 300 bp to 1,000 bp, respectively, were constructed and sequenced on the Illumina HiSeq 2,000 platform to obtain low error short reads. For nanopore sequencing, 2 µg of genomic DNA was end-repaired (NEBNext Ultra II End Repair Kit, New England Biolabs, MA, United States) and cleaned with ×1 AMPure beads (Beckman Coulter, United States). NEB blunt/TA ligase (New England Biolabs, MA, United States) was used to perform adapter ligations (AMX) for 30 min. Library mix was cleaned up using 0.4× AMPure beads (Beckman Coulter, United States) and finally eluted in 16 µl of elution buffer. A total of 480 ng of sequencing library was obtained and used for sequencing. Long reads were obtained by sequencing on MinION MklB (Oxford Nanopore Technologies, Oxford, United Kingdom) using spot on flow cell (R9.4), and base calling was performed using Metrichor Nanopore. A total of 46.2 Gb of raw data was generated on the Oxford Nanopore and Illumina platforms.

End-repairing of DNA sample of 2019SD1 was performed using NEBNext Ultra II End Repair kit (NEW ENGLAND BIOLABS, MA, USA) and subsequent clean up with 1 × Ampure beads (BECKMANN COULTER, USA). Thereafter, native barcode ligation was carried out with NEB Blunt/TA ligase (NEW ENGLAND BIOLABS, MA, USA) using nbd103 (ONT) and cleaned with 1 × Ampure beads. Further, the barcode-ligated DNA sample was quantified using Qubit 4 (THERMOFISHER SCIENTIFIC, USA). In subsequent step, bam adapter ligation was performed for 15 min using NEBNext quick ligation module (NEW ENGLAND BIOLABS, MA, USA). Again, the library mix was cleaned up with the help of 0.4 × Ampure beads (BECKMANN COULTER, USA). Finally, the sequencing library was eluted in 15 µL of elution buffer and used for Nanopore sequencing. Gridion × 5 (OXFORD NANOPORE TECHNOLOGIES, OXFORD, UK) with spoton flow cell (r9.4) was used for sequencing in a 48 h sequencing protocol on Minknow 2.1 v18.05.5. In order to eliminate probable errors in long-read assemblies, nanopore raw reads (‘fast5’ format) were basecalled (‘fastq5’ format) and demultiplexed using Albacore v2.3.1. Further for sequence polishing, basecalled reads were error-corrected and assembled using “Canu” assembler v1.8.

End-repairing of bacteriophage HCF1 DNA samples was done using NEBnext ultra II end repair kit (New England Biolabs, Ipswich, MA, United States). Clean-up of end-repaired samples was carried out with 1x AmPure beads (Beckmann Coulter, United States). NEB blunt/TA ligase (New England Biolabs) using NBD103 (ONT) was used for Native barcode ligation. Following ligation, clean-up process was repeated with 1x AmPure beads (Beckmann Coulter). The Qubit-quantified barcode ligated DNA sample was then pooled at equimolar concentrations to obtain 500 ng pooled sample. BAM Adapter ligation was performed for 15 min using NEBnext Quick Ligation Module (New England Biolabs). The library mix was cleaned up using 0.4X AmPure beads (Beckmann Coulter) and, finally, the sequencing library was eluted in 15 μL of elution buffer and used for Nanopore sequencing. Sequencing was performed on a GridION X5 (Oxford Nanopore Technologies, Oxford, United Kingdom) using a SpotON flow cell (R9.4) in a 48 h sequencing protocol on MinKNOW 2.1 v18.05.5. In order to eliminate probable errors in long-read assemblies, all the Nanopore raw reads (“fast5” format) were basecalled (“fastq5” format) and demultiplexed using Albacore v2.3.1. Basecalled reads were error-corrected and assembled using “Canu” assembler v1.8, for sequence polishing.

For whole genome metagenome sequencing, 300 ng of genomic DNA was used after end-repairing (NEBnext ultra II end repair kit, New England Biolabs, MA, United States) and cleaning up with 1× AmPure beads (BeckmannCoulter, United States). The DNA samples were barcoded (LongAmp Taq 2× New England Biolabs, MA, United States) and cleaned up with 1.6× AmPure beads (Beckmann-Coulter, United States). The end-repairing was performed using NEBnext (New England Biolabs, MA, United States) and adapter ligation was performed for 10 min using NEB blunt/TA ligase (New England Biolabs, MA, United States). Library mix was cleaned up using 0.6× AmPure beads and finally eluted in 15 μl of elution buffer. The processed DNA samples were sequenced on GridION X5 (Oxford Nanopore Technologies, Oxford, United Kingdom) using SpotON flow cell (R9.4) in a 48 h sequencing protocol on MinKNOW 2.1 v18.05.5. Nanopore raw reads (“fast5” format) were base called (“fastq5” format) and demultiplexed using Albacore v2.3.1. The reads were compared against NCBI nr database using the diamond tool. The diamond BLASTX alignments were further converted to MEGAN readable format by using the NCBI taxonomy to summarize and order the results. MEGAN GUI is then used to estimate and interactively explore the taxonomical content by checking the read assignment from phylum to species level classification.