Additional file 1: of OPERA-LG: efficient and exact scaffolding of large, repeat-rich eukaryotic genomes with performance guarantees

Contains supplementary notes, figures and tables. (DOCX 2996 kb

MOESM1 of Development of a genetically programed vanillin-sensing bacterium for high-throughput screening of lignin-degrading enzyme libraries

Additional file 1: Figure S1. Determination of sub-lethal dose of vanillin on E. coli BL21 cells; Figure S2. Cluster heat map showing global gene expression correlation between different sets of samples used in RNAseq experiments; Figure S3. Schematic diagram showing location of putative promoter region and the plasmid constructs for development of the whole-cell biosensor; Table S1. Top seven up-regulated genes of E. coli BL21 strain after exposure to sublethal dose of vanillin and function of the encoded proteins; Table S2. Assayed promoter sequences from top seven up-regulated genes; Table S3. Fluorescence of the 5 mM vanillin induced live cell biosensors (Lcb1–Lcb7)

Evaluation of EMIRGE, modQIIME and RTAX on different datasets.

<p>Precision and recall rates for the “Oral”, “Gut”, “Complex” and ABC33 datasets using EMIRGE, modQIIME and RTAX at a 0.1% relative abundance threshold. The percentage of sequences/OTUs removed because of the abundance threshold is given in parentheses for each method.</p

<i>In silico</i> evaluation of 16S rRNA PCR primers.

<p>A) Percentage of sequences matching individual primers, with the top two primers highlighted in boxes. B) Percentage of sequences amplifiable by various primer pairs (338F*/1061R is the best pair). Percentage of matched sequences is measured against the Greengenes 16S rRNA sequence database. See Table S4 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060811#pone.0060811.s001" target="_blank">File S1</a> for primer sequences and results measured against the RDP and SILVA databases. Primer numbering is based on the <i>E. coli</i> system of nomenclature as in Brosius <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060811#pone.0060811-Brosius1" target="_blank">[37]</a> and for simplicity the same name (say 784F) is used for both forward and reverse primers at a given position.</p

Species- and genus-level resolution of various sequencing approaches.

<p>Resolution was measured by the number of OTUs/clusters produced using UCLUST <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060811#pone.0060811-Edgar1" target="_blank">[21]</a> at the species (97% identity) and genus level (95% identity) for 16S rRNA sequences in the Greengenes database, based on various end-sequencing (76 bases in length from either the 5′ or 3′ end) and shotgun-sequencing approaches and primer combinations. A higher OTU/cluster number indicates a theoretical higher level of resolution for taxonomic classification. The numbers in parenthesis provide the purity of clusters as measured by the percentage of clusters with homogenous taxonomy assignments in Greengenes. Entries with the highest resolution and/or purity for each sequencing approach are marked in bold. The primer sequences can be found in <b>Table S4</b> in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060811#pone.0060811.s001" target="_blank">File S1</a></b>.</p

Community composition based on 16S rRNA sequence reconstruction using EMIRGE.

<p>A) Correlation between known and estimated relative abundances of predicted species on three <i>in silico</i> datasets. A log-scaled version of this plot can be seen in <b>Figure S1</b> in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060811#pone.0060811.s001" target="_blank">File S1</a></b>. B) Composition at the phylum level for the throat swab and stool sequencing datasets.</p

Measurement of fetal fraction in cell-free DNA from maternal plasma using a panel of insertion/deletion polymorphisms

<div><p>Objective</p><p>Cell-free DNA from maternal plasma can be used for non-invasive prenatal testing for aneuploidies and single gene disorders, and also has applications as a biomarker for monitoring high-risk pregnancies, such as those at risk of pre-eclampsia. On average, the fractional cell-free fetal DNA concentration in plasma is approximately 15%, but can vary from less than 4% to greater than 30%. Although quantification of cell-free fetal DNA is straightforward in the case of a male fetus, there is no universal fetal marker; in a female fetus measurement is more challenging. We have developed a panel of multiplexed insertion/deletion polymorphisms that can measure fetal fraction in all pregnancies in a simple, targeted sequencing reaction.</p><p>Methods</p><p>A multiplex panel of primers was designed for 35 indels plus a <i>ZFX/ZFY</i> amplicon. cfDNA was extracted from plasma from 157 pregnant women, and maternal genomic DNA was extracted for 20 of these samples for panel validation. Sixty-one samples from pregnancies with a male fetus were subjected to whole genome sequencing on the Ion Proton sequencing platform, and fetal fraction derived from Y chromosome counts was compared to fetal fraction measured using the indel panel. A total of 157 cell-free DNA samples were sequenced using the indel panel, and informativity was assessed, along with the proportion of fetal DNA.</p><p>Results</p><p>Using gDNA we optimised the indel panel, removing amplicons giving rise to PCR bias. Good correlation was found between fetal fraction using indels and using whole genome sequencing of the Y chromosome (Spearmans r = 0.69). A median of 12 indels were informative per sample. The indel panel was informative in 157/157 cases (mean fetal fraction 14.4% (±0.58%)).</p><p>Conclusions</p><p>Using our targeted next generation sequencing panel we can readily assess the fetal DNA percentage in male and female pregnancies.</p></div

Frequencies of maintained SNVs compared to SNVs lost during horizontal transmission.

<p><b>(A)</b> Frequencies of DENV2 SNVs maintained from human to mosquito, versus those present only in the human. <b>(B)</b> Frequencies of DENV2 SNVs maintained from mosquito abdomen to mosquito salivary gland, versus those present only in the abdomen. <b>(C)</b> Frequencies of SNVs maintained in one mosquito per human-mosquito group, versus those maintained in more than 1 mosquito per group. Boxplots indicate medians and 25th and 75th percentiles; whiskers indicate minimum and maximum values. *, p < 0.05; ***, p < 0.0001; Mann-Whitney test.</p

Selection pressures on the DENV genome, analyzed per gene, at different stages of horizontal transmission.

<p>Ratios of the number of non-synonymous (NS) to synonymous (S) SNVs per gene are shown for <b>(A)</b> human- versus mosquito-derived (both abdomen and salivary gland) DENV populations, and <b>(B)</b> mosquito abdomen- versus salivary gland-derived DENV populations. Human-derived samples are a pool of DENV2 patient samples from this study and DENV1 and DENV3 patient samples from the EDEN study [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004052#pntd.0004052.ref019" target="_blank">19</a>]. Numbers of NS and S SNVs for each gene are indicated in tables below the graphs. *, p < 0.05; Fisher's exact test (M, p = 0.00439; E, p = 0.031; NS1, p = 0.002).</p

Loss and maintenance of SNVs during horizontal transmission.

<p><b>(A)</b> Percentage of SNVs maintained during transmission from human to mosquito and from mosquito abdomen to mosquito salivary gland. <b>(B)</b> Tracking of maintained SNVs. The number of instances of each scenario are shown; these include all occurrences of SNVs that were found to be maintained in more than one mosquito. <b>(C)</b> Percentage of SNVs maintained between members of a human-human transmission pair from the EDEN study.</p