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Additional file 4: Table S4. Differentially expressed proteins between “P0” and “F3”

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Additional file 2: Table S2. Composition of total raw reads and statistical results of samples mapped to gene and genome

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Additional file 1: Table S1. Primers used in this study

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Additional file 3: Table S3. Differentially expressed genes between “P0” and “F3”

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Additional file 7: Figure S1. Comparative metabolomic analyses of R. toruloides samples prepared under Pi-limited (P0) and Pi-replete (F3) conditions. a Total ion chromatogram of quality control (QC) sample (positive). b Total ion chromatogram of QC sample (negative). c Changes of some cellular nucleoside derivatives and bases. Figure S2. Metabolomic analysis of R. toruloides during phosphate-limitation. a Score plot of PCA in P0 vs F3 (positive). b Score plot of PLS-DA in P0 vs F3 (positive). c Sorting plot of PLS-DA in P0 vs F3 (positive). d Score plot of OPLS-DA in P0 vs F3 (positive). Figure S3. Metabolomic analysis of R. toruloides during phosphate-limitation. a Score plot of PCA in P0 vs F3 (negative). b Score plot of PLS-DA in P0 vs F3 (negative). c Sorting plot of PLS-DA in P0 vs F3 (negative). d Score plot of OPLS-DA in P0 vs F3 (negative)

A Novel Multiplex Tetra-Primer ARMS-PCR for the Simultaneous Genotyping of Six Single Nucleotide Polymorphisms Associated with Female Cancers

<div><p>Background</p><p>The tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) is a fast and economical means of assaying SNP's, requiring only PCR amplification and subsequent electrophoresis for the determination of genotypes. To improve the throughput and efficiency of T-ARMS-PCR, we combined T-ARMS-PCR with a chimeric primer-based temperature switch PCR (TSP) strategy, and used capillary electrophoresis (CE) for amplicon separation and identification. We assessed this process in the simultaneous genotyping of four breast cancer–and two cervical cancer risk–related SNPs.</p><p>Methods</p><p>A total of 24 T-ARMS-PCR primers, each 5′-tagged with a universal sequence and a pair of universal primers, were pooled together to amplify the 12 target alleles of 6 SNPs in 186 control female blood samples. Direct sequencing of all samples was also performed to assess the accuracy of this method.</p><p>Results</p><p>Of the 186 samples, as many as 11 amplicons can be produced in one single PCR and separated by CE. Genotyping results of the multiplex T-ARMS-PCR were in complete agreement with direct sequencing of all samples.</p><p>Conclusions</p><p>This novel multiplex T-ARMS-PCR method is the first reported method allowing one to genotype six SNPs in a single reaction with no post-PCR treatment other than electrophoresis. This method is reliable, fast, and easy to perform.</p></div

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Additional file 5: Table S5. Model summaries for the discrimination between the Pi-replete and Pi-limited samples from LC-MS data for metabolomic analysis

Primers for the multiplex tetra-primer ARMS-PCR for SNPs associated with breast and gynecologic cancers.

a<p>Underlined is the universal tag sequence at the 5′ end of the chimeric primers.</p>b<p>Allele-specific nucleotides at the 3′ end are indicated in bold letters.</p>c<p>Specificity is increased by the introduction of a deliberate mismatch at position -1 of the polymorphism site, indicated by bold lower case letters.</p>d<p>A pair of universal primers annealing to the 5′ portion of each chimeric primer.</p

Direct sequencing result of sample #1101–291(corresponding to the sample in lane 1, Fig.1B), which showed the combination of 5 heterogenous and 1 homogenous SNPs.

<p>Direct sequencing result of sample #1101–291(corresponding to the sample in lane 1, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062126#pone-0062126-g001" target="_blank">Fig.1B</a>), which showed the combination of 5 heterogenous and 1 homogenous SNPs.</p

Genotypes and allele frequencies of tested samples in this study.

a<p>increased risk allele in bold</p>b<p><i>p</i> value was calculated using chi test</p