SMRT sequencing results summary.

<p>^§as estimated by gel electrophoresis of cloned expansion fragment excised from plasmid backbone.</p><p>^<b>¶</b><b>As compared with Sanger sequencing.</b></p><p>*Alignment based on reference genome sequence (NC_000022.11; GI: 568815576, Region: 45794523..45796589) using LALIGN.</p><p>^as determined by counting motif blocks in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135906#pone.0135906.g002" target="_blank">Fig 2</a>. Rpts, repeats; nts, nucleotides; bp, base pairs</p><p>SMRT sequencing results summary.</p

pJAZZ-OCmin vector.

<p>The telN gene and several non-essential genes from phage N15 (purple boxes) were deleted from the vector pJAZZ-OC. repA, cB: replication protein genes; Cam^r, chloramphenicol resistance; T, terminator. Closed terminal hairpin structures are indicated by black circles.</p

Proportion of repeat motifs in SCA10 expansions.

<p>Proportions are calculated as the percentage of nucleotides of each motifs divided by the total number of nucleotides for each expansion. Motifs present in SMRT sequence results that are verified by Sanger sequencing methods (“shotgun”) comprise the majority of motifs seen (A) while some motifs are unverified (B). Green, SMRT sequencing results from subject A; Green hatched, random shotgun sequencing results from subject A; Blue, SMRT sequencing results from subject B; Red, SMRT sequencing result from subject C; Red hatched, random shotgun sequencing results from subject C.</p

Schematic representations of the repeat expansions.

<p>(A) SCA10 expansion in subject A. (B) SCA10 expansion in subject B. (C) SCA10 expansion in subject C. Rectangles represent sequence motifs, as indicated by the color key, in the 5’ (upper left) to 3’ (lower right) direction. Black rectangles indicate unverified motifs described further in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135906#pone.0135906.g003" target="_blank">Fig 3B</a> and are indicated as follows: A: ATTTCT, ATTTCT; B: ATTTCT, A, ATTTCT, A, ATTCCT, TAC, ATTTCT, A, ATT, ACTTCT, ATTCA, ATTTCT, ATTTCT, T, ACTTTCT, TCTTTCT, ATTT, ATTTCT, ATCT, ATTTCT, ATTTCT, ATTTCT, ATTTCT, ATTTCT, T, ATCC, ATTC, ATTTCC, C, ATTTCC, TTCCC, ATTTCC, CATCC, ATTTCC, C, C, C, C, ATTC, ATTTCC, ATTCC; C: ATCT, ATCT, ATCT, AT, ATCT, T, ATC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, C, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, ATC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, C</p

SCA10 expansion templates for SMRT sequencing.

<p>(A) PCR amplification of the SCA10 expansion from gDNA extracted from blood lymphocytes (Subjects A and B) or from somatic cell hybrid lines (subject C). Lanes are cropped from non-adjacent lanes of the same gel. The full gel is shown is <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135906#pone.0135906.s001" target="_blank">S1 Fig</a>. Arrows indicate the size of bands that were excised for cloning and sequencing (subject A, the 6.5 kb band; subject B, the 5.9 kb band; subject C, the 4.7 kb band) (B) Purified template from cloned PCR products in Fig 1A for SMRT sequencing. L: 1 kb ladder (New England Biolabs). The full gels are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135906#pone.0135906.s002" target="_blank">S2 Fig</a>.</p

ExCyto PCR amplification.

<p>A cDNA library was transformed into ExCyto cells. Twenty-four different cDNAs were amplified without addition of exogenous polymerase using (A) single colonies, (B) 2 µl of frozen glycerol stocks, or (C) 2 µl of overnight cultures. One kb molecular weight markers are shown in the last lane.</p

Dilution series of ExCyto PCR cells.

<p>Eight different clones from a cDNA library transformed into ExCyto cells were grown overnight, and a serial dilution was used for amplification (undiluted, 100-, and 1000-fold dilutions). In (A), bacteria were diluted, reducing both the target DNA and the tsDNA polymerase. In (B), to compensate for the serial dilution of the tsDNA polymerase, 2 ul of ExCyto cells (OD 1.1) without plasmids was added. In (C), to compensate for the serial dilution of plasmid, 2 ul of bacteria with the same plasmid, but without the tsDNA polymerase was added. A 10-fold dilution is not shown, but gives similar amplification to that seen with undiluted cells. One kb molecular weight markers are shown in the last lane.</p

ExCyto PCR of chromosomal integrants.

<p>Two µl of overnight cultures from chromosomal integrants were used for PCR with primers flanking the melAmelB bicistron integration site. Integrants show a band at 4.5 kb. Wild type (WT, last lane) has a band at 2 kb. One kb molecular weight markers are shown in the first lane.</p