Fig 1.
Agarose gel electrophoresis of T7 SDA reactions.
(A) Amplicons produced in the reaction of λ DNA with a NEase and either the WT or Δ28 gp5 T7 replisome. (B) A scheme for DNA amplification of a region between two NEase recognition sites. (C) Illustration of NEase recognition sites within the linear λ phage genome. Vertical lines above the horizontal correspond to nicks on the top strand of DNA, and those below to nicks on the bottom strand of DNA. Shaded regions correspond to amplicons.
Table 1.
Nanopore sequencing statistics for T7 SDA reactions.
Fig 2.
Coverage maps from nanopore sequencing data for T7 SDA reactions.
Each panel shows 3’, 5’ and full coverage for all alignments produced by minimap2 including those from chimeric reads. Counts per total alignments reflect the coverage at each nucleotide divided by the number of alignments. At top are diagrams showing nicking recognition sites within the λ phage genome, where lines above the horizontal correspond to anticipated top strand nicks, and those below to bottom strand nicks. The nicking enzyme and polymerase used in each panel are (A) Nt.BbvCI and Δ28 gp5; (B) Nb.BbvCI and Δ28 gp5; (C) Nb.BssSI and Δ28 gp5; (D) Nt.BbvCI and WT gp5; (E) Nb.BbvCI and WT gp5; and (F) Nb.BssSI and WT gp5.
Fig 3.
Identification of dsDNA amplicons in a T7 SDA reaction.
Data are shown for the reaction of Δ28 gp5 and Nt.BbvCI. (A) Kernel density estimate plot of read length against alignment length. Reads were filtered to have average basecall quality greater than 10. Only one alignment is considered per read. (B) Coverage maps of reads with specified lengths. At top is a diagram showing recognition sites for Nt.BbvCI in the λ phage genome. Two additional sites of sequence GCTTAGG are denoted with asterisks.
Fig 4.
Amplification of hairpins in a T7 SDA reaction.
Data are shown for the reaction of WT gp5 and Nt.BssSI. (A) Kernel density estimate (KDE) plot of read length against alignment length. Only one alignment is considered per read. (B) KDE plot of read length against average read quality score. (C) Non-denaturing agarose gel of the reaction products stained with ethidium bromide. (D) Denaturing alkaline agarose gel of the reaction products. In each panel, clusters of amplified hairpin DNA molecules are labeled with lower case letters.
Fig 5.
Inverted repeats are sites for template switching.
(A) 3’ Coverage over the region 8695–8649 in the reaction with Nb.BbvCI. Strand displacing polymerization may only be initiated from the right. (B) 3’ Coverage over the region 40580–40634 in the reaction with Nb.BssSI. There are nicking recognition sequences on both sides of this region, but polymerization is principally initiated from the left. (C) Selected reads showing template switching. Representations of the full alignments for selected reads are shown in S5 Fig in S1 File.
Fig 6.
A scheme for amplification of hairpin DNA.
(I) Replisome dissociation and DNA branch migration produce a cruciform structure at a suitable inverted repeat sequence, depicted as red and blue blocks. (II) 3’ exonuclease activity of WT gp5 enables extension of the cruciform intermediate. Extension may restart at either the first or second instance of the inverted sequence. (III) Another round of nicking allows for hairpin release. Hairpin release may be assisted by another round of polymerization from the DNA nick.