Klenow fragment dna polymerase

RP11-242C23 was digested using EcoRI and treated with Klenow Fragment DNA Polymerase (Takara, Shiga, Japan) at 37 °C for 20 min. DNA was subjected to electrophoresis on a 0.5% agarose gel. Bands larger than the 10-kb marker (GeneRuler 1 kb DNA Ladder; Thermo Fisher Scientific, Waltham, MA, USA) were excised using a razor under ultraviolet light. The DNA fragments larger than 1 kb were subjected to phenol-chloroform DNA preparation. Agarose gels were soaked in phenol and incubated for 30 min at −80 °C. Then, the aqueous phase was collected and phenol-chloroform DNA preparation was performed. The EcoRI-digested whole D4Z4 repeat was enriched in the DNA sample.

These repeat containing plasmids were cut (linearized) with restriction enzymes NheI, EcoRI-HF, BamHI-HF, or DraIII (NEB, MA, USA) (Additional file 1: Table S7) and then treated with Klenow Fragment DNA Polymerase (Takara, Shiga, Japan) at 37 °C for 30 min. The whole DNA fragments were purified using AmPureXT beads (Agilent Technologies, CA, USA), then subjected to nanopore sequencing. Library preparation was performed using a 1D native barcoding genomic DNA kit (EXP-NBD103 and SQK-LSK108) and then subjected to MinION (Oxford Nanopore Technologies) sequencing using one FLA-MIN106 (R9.4.1) flow cell according to the manufacturer’s protocol. Basecalling and fastq conversion were performed with MinKNOW ver1.11.5. De-barcoding was done using EPIME software (Oxford Nanopore Technologies).
Obtained fastq files were transformed to fasta files using seqkit fq2fa option (http://bioinf.shenwei.me/seqkit). fasta files were aligned to plasmid references like this:

lastdb -P8 -uNEAR -R01 plasmid-ref plasmid.fasta

last-train -P8 plasmid-ref reads.fasta > train.out

lastal -P8 -p train.out plasmid-ref reads.fasta | last-split > alns.maf