Uniquely and commonly called SNPs in TruSeq Nano and NEBNext Ultra-prepared isolates.

<p>The majority of called SNPs were common to both methods in each isolate. However, a greater number of SNPs were unique to the isolate prepared with the TruSeq Nano method (blue). Venn diagrams were generated using the Venny software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113501#pone.0113501-VENNY1" target="_blank">[17]</a> of SNPs called using GATK <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113501#pone.0113501-VanderAuwera1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113501#pone.0113501-DePristo1" target="_blank">[15]</a>.</p

False positive rates associated with called INDELs against the <i>Cng</i> reference [9].

<p>False positive rates associated with called INDELs against the <i>Cng</i> reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113501#pone.0113501-Loftus1" target="_blank">[9]</a>.</p

Cost comparison for library prep consumables, based on UK list prices (May 2014) where possible.

<p>Quality control analysis included quantification of all samples by Qubit Broad Range dsDNA assay (Life Technologies) prior to beginning, follow by final analysis using TapeStation 2200 D1K Screen Tapes (Agilent) and qPCR using the Kapa kit for Illumina libraries (Kapa Biosciences). Filter tip and Ampure XP bead costs are based on estimates of usage, with ‘per sample’ usage rounded up to the nearest tip.</p><p>Cost comparison for library prep consumables, based on UK list prices (May 2014) where possible.</p

IQR of read depths of TruSeq Nano and NEBNext Ultra prepared samples.

<p>IQR of read depths of TruSeq Nano and NEBNext Ultra prepared samples.</p

Coverage is biased at AT- and high GC-rich regions.

<p>Normalised coverage (binned into 100 bp windows) relating to GC content, where the blue line represents the TruSeq Nano-prepared isolate, and the yellow line represents the NEBNext Ultra-prepared isolate. The black dotted line at <i>x</i> = 1 is the expected normalised coverage showing no bias. Whilst both library preparation methods perform similarly, the TruSeq Nano-prepared isolates generally provide more coverage at AT-rich regions.</p

SNP calls from two different pipelines and false positive rates associated with calling SNPs against the <i>Cng</i> reference [9].

<p>SNP calls from two different pipelines and false positive rates associated with calling SNPs against the <i>Cng</i> reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113501#pone.0113501-Loftus1" target="_blank">[9]</a>.</p

Phylogenetic and Bayesian analysis of concatenated nucleotide sequences.

<p>(A) SplitsTree neighbour network shows diverse VNII and VNB, and clonal VNI clades. (B) Analysis of VNI clustering using StructureHARVESTER and ΔK shows optimal number of K clusters is 3. (C) Use of STRUCTURE allows VNI sequences to be subdivided into 3 distinct populations: VNI(a), VNI(b), VNI(c).</p

Median <i>in vitro</i> phenotyping values ordered by molecular type, VNI Subtype and high frequency MLST.

<p>Kruskal Wallis analysis performed on groups, and p values shown. Overall median is plotted in red. (A) Survival in <i>ex vivo</i> CSF, (B) Laccase Activity normalised to H99 reference strain, (C) <i>In vitro</i> phagocytosis of isolates by J774 cells (per 1 μl lysate).</p

Clinical characteristics and survival of patients infected with different lineages of <i>C neoformans</i>.

<p>(A) Patient survival by molecular type; VNB isolates (n = 8) were associated with significantly worse outcome compared to VNI and VNII isolates at 70 days (p = 0.01) and 365 days (p = 0.003) follow-up in Cox’s proportional hazard survival analysis. (B) Patient survival by high frequency MLST type. (C) Number of patients with and without altered mental status by molecular group (% with altered mental status indicated above each bar). (D) Baseline fungal burden, showing variation by high frequency MLST type.</p

<i>In vitro</i> phenotyping results ordered by molecular type, VNI subtype, and high frequency MLST type.

<p>* CSF survival was not performed for one of the eight VNB isolates.</p><p><i>In vitro</i> phenotyping results ordered by molecular type, VNI subtype, and high frequency MLST type.</p