Searching for non-B DNA-forming motifs using nBMST (non-B DNA motif search tool)

Abstract

This unit describes basic protocols on using the non-B DNA Motif Search Tool (nBMST) to search for sequence motifs predicted to form alternative DNA conformations that differ from the canonical right-handed Watson-Crick double-helix, collectively known as non-B DNA, and on using the associated PolyBrowse, a GBrowse-based genomic browser. The nBMST is a Web-based resource that allows users to submit one or more DNA sequences to search for inverted repeats (cruciform DNA), mirror repeats (triplex DNA), direct/tandem repeats (slipped/hairpin structures), G4 motifs (tetraplex, G-quadruplex DNA), alternating purine-pyrimidine tracts (left-handed Z-DNA), and A-phased repeats (static bending). The nBMST is versatile, simple to use, does not require bioinformatics skills, and can be applied to any type of DNA sequences, including viral and bacterial genomes, up to an aggregate of 20 megabasepairs (Mbp).

Figure 1

The nBMST submission page. The 5 steps involved in the submission process are shown. An email address is entered and all the non-B DNA motifs are selected (grayed area). A FASTA sequence, NC_007530.2 Bacillus anthracis str. ‘Ames Ancestor’, in this example is uploaded. The captcha characters (4AkA4 in this instance) are entered since the user did not log in.

Figure 2

The nBMST results page. The upper section includes the major statistics of the nBMST run and the lower section displays expandable individual results for each motif type.

Figure 3

Static visualization of the direct repeats as a PNG image. The PNG image may be saved either by right-clicking on it or by clicking on “Download all files for this motif” on the upper right.

Figure 4

Dynamic visualization of the direct repeats on PolyBrowse page. The PolyBrowse page is created uniquely for each nBMST job submitted and is visible only to the user who submitted the job. In this example, job ID 8c32923d93, a 5 Mbp region of the Bacillus anthracis results is displayed here.

Figure 5

A smaller region, 6.001 kbp, of Figure 4 is zoomed in and all the non-B DNA motif tracks are turned on. The −/+ sign (left of the motif tracks) may be used to toggle between hiding and showing the results.

Figure 6

Dynamic visualization of multiple FASTA files. When multiple FASTA sequences are submitted, PolyBrowse displays multiple sub-links (red box), each representing a specific sequence. Clicking on each sub-link displays the non-B DNA motifs found for that specific sequence.

Figure 7

PolyBrowse general page layout displaying File and Help options, the Landmark or Region where coordinates of around human c-MYC gene is entered and Human v37 selected as Data Source. Also shown are Gene tracks including Refseq Genes, mRNAs and Promoters; Non-B DNA motif track G-Quadruplex Forming Repeat and Polymorphism track dbSNP. Note that out of the three G-quadruplex repeat motifs (shown in blue glyph), two are predicted within the promoter, MYC_Prom (orange glyph) and one within the first exon of mRNA, NM_002467.3 (gray glyph) of the MYC gene. One dbSNP entry, rs13250910 (red glyph), falls within one G-quadruplex motif.

Figure 8

PolyBrowse page showing Non-B DNA motifs section where annotation track for G-Quadruplex Forming Repeat is turned on. Another track turned on is 1k LiftOver Blocks from Synteny section.

Figure 9

PolyBrowse page showing “trace GPlexes clusters” tracks where “trace” refers to Sanger trace reads which have been mapped against the human genome reference (version 37.1).

Figure 10

PolyBrowse. 1k LiftOver blocks available for cross species comparison. Note that when chr8_128748001_LO1k is hover upon, the syntenic regions for other species are displayed. Clicking on a new species takes the user to the selected species. In the example in Figure 13, we clicked on

Figure 11

PolyBrowse page showing the MYC gene in Chimp v2 as Data Source. Note that three G-quadruplex forming repeat motifs are all conserved in the chimpanzee genome sequence. Similar to human, out of the three G-quadruplex repeat motifs (shown in blue glyph), two are predicted within the promoter, MYC_Prom (orange glyph) and one within the first exon of mRNA, XM_519958.2 (gray glyph) of the MYC gene.

Figure 12

The 16 and 17 bp G4 DNA-forming structures reported in Cahoon and Seifert (2009) are captured by nBMST. (A) Details of the G4 motifs in the Neisseria gonorrhoeae genome NCCP11945 (B) Details of the G4 motifs in the Neisseria gonorrhoeae genome FA 1090 (C) The genomic region of NCCP11945 containing both the 16 and 17 bp G4 DNA-forming repeats as seen in PolyBrowse (D) The details of the 17 bp G4 DNA-forming repeat is obtained by clicking on the green track in (C).

Figure 13

Non-B DNA-forming motifs detected by nBMST in the study by Lawson et al. (2011). (A) Nucleotide sequence of the 67 bp de novo insertion observed in patient PA27. (B) PolyBrowse view of the nBMST results for the 220 bp sequence from patient PA27 containing the 67 bp de novo insertion which spans from 62 to 128 nt. Most of the motifs occurred near or within the 67-bp de novo insertion.