Additional file 2: Table S2. of Identification of candidate protective variants for common diseases and evaluation of their protective potential

Comparison of methods (PolyPhen-2 and CADD) to predict functional effect of CPVs. (DOCX 92 kb

Mapped small RNAs in <i>E</i>. <i>invadens</i> show relatively greater occupancy of promoter regions compared to <i>E</i>. <i>histolytica</i>.

<p>Small RNA sequences from <i>E</i>. <i>histolytica</i> and <i>E</i>. <i>invadens</i> trophozoites were aligned to the region from -100 to +100 (relative to ATG) of each annotated ORF. The frequency of antisense RNA 5' ends (for genes with ≥20 antisense RNAs) at each position was plotted. The ratio of small RNAs in the upstream region (-100 to ATG) to small RNAs in the coding region (ATG to +100) of each ORF is shown.</p

Additional file 1: Table S1. of Identification of candidate protective variants for common diseases and evaluation of their protective potential

Summary of filtering stages performed on GWAS catalog. (DOCX 59 kb

A custom-made antibody to <i>Entamoeba</i> AGO2-2 detects a 110kDa protein, which is enriched in the nucleus.

<p><b>(A)</b> Western Blot detects a band at 110 kDa using α-EhAGO2-2 antibody on <i>E</i>. <i>invadens</i> whole cell lysate, cytosolic fraction and nuclear fractions. <b>(B)</b> Immunofluorescence assay on <i>E</i>. <i>invadens</i> trophozoites showed nuclear enriched signal, which co-localizes with DAPI. Some cytoplasmic signal is also noted.</p

Composition of small RNA libraries from <i>E</i>. <i>histolytica</i> under basal and stress conditions.

<p>Sequenced libraries from each condition were processed to remove barcodes, size selected for reads >15nt or <40nt, duplicate reads removed and the remaining unique reads sequentially mapped against each sequence type shown. Note that a small number of reads mapped both antisense and sense to transcripts, hence the total number of reads mapped to “transcript” is lower than the total number of reads mapping antisense plus sense. Total number of reads and percent of unique mapped reads are shown.</p

Comparison of genes with abundant small RNAs during <i>E</i>. <i>invadens</i> development.

<p><b>(A)</b> Venn diagram of genes with ≥20 AS small RNAs mapped at each time-point. Note that the majority of genes with small RNAs are represented in all time points. Due to the smaller number of reads in the trophozoite library, all genes with ≥20 small RNAs in that library are present in the other libraries; hence this time point was not included in the diagram. <b>(B)</b> Genes with ≥20 AS small RNAs are not expressed during development. Expression data are based on RNA-Seq from <i>E</i>. <i>invadens</i> trophozoites, early cysts (24h), mature cysts (72h) and excysting parasites are shown as Fragments per kilobase per million mapped reads (FPKM). The 789 genes to which ≥20 AS small RNAs mapped in any timepoint had very low expression under all timepoints (red), compared to expression of all genes (blue), indicating that they are not regulated during development.</p

Additional file 1 of Illumina error profiles: resolving fine-scale variation in metagenomic sequencing data

Data further supporting the results including additional figures (Figures S1-S18) and tables (Tables S1-S3). (PDF 2808 kb

Mapping of <i>E</i>. <i>invadens</i> small RNAs to transposons and repetitive elements.

<p>Unique reads from <i>E</i>. <i>invadens</i> trophozoites, after removal of tRNA and rRNA mapped reads, were aligned to each type of transposable and repetitive element found in the <i>E</i>. <i>invadens</i> genome. Total read number, percent of unique reads, and percent of the <i>E</i>. <i>invadens</i> genome that each element represents (adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134481#pone.0134481.ref030" target="_blank">30</a>]) are shown.</p

<i>E</i>. <i>invadens</i> small RNA length and nucleotide distribution.

<p>After removal of duplicates and size selection to remove RNAs >15nt or <40nt, a profile of length distribution and percent of each nucleotide base at each position were calculated for each library. Note the strong peak at 27nt in length and the preference for G at the 5' end in both datasets. (<b>A)</b> Trophozoite library, and (<b>B)</b> Mature cyst (72h) library.</p

High Throughput Sequencing of <i>Entamoeba</i> 27nt Small RNA Population Reveals Role in Permanent Gene Silencing But No Effect on Regulating Gene Expression Changes during Stage Conversion, Oxidative, or Heat Shock Stress

<div><p>The human parasite <i>Entamoeba histolytica</i> has an active RNA interference (RNAi) pathway with an extensive repertoire of 27nt small RNAs that silence genes. However the role of this pathway in regulating amebic biology remains unknown. In this study, we address whether silencing via 27nt small RNAs may be a mechanism for controlling gene expression changes during conversion between the trophozoite and cyst stages of the parasite. We sequenced small RNA libraries generated from trophozoites, early cysts, mature cysts, and excysting cells and mapped them to the <i>E</i>. <i>invadens</i> genome. Our results show that, as in <i>E</i>. <i>histolytica</i>, small RNAs in <i>E</i>. <i>invadens</i> are largely ~27nt in length, have an unusual 5'-polyphosphate structure and mediate gene silencing. However, when comparing the libraries from each developmental time-point we found few changes in the composition of the small RNA populations. Furthermore, genes targeted by small RNAs were permanently silenced with no changes in transcript abundance during development. Thus, the <i>E</i>. <i>invadens</i> 27nt small RNA population does not mediate gene expression changes during development. In order to assess the generalizability of our observations, we examined whether small RNAs may be regulating gene expression changes during stress response in <i>E</i>. <i>histolytica</i>. Comparison of the 27nt small RNA populations from <i>E</i>. <i>histolytica</i> trophozoites from basal conditions, or after heat shock or exposure to oxidative stress showed few differences. Similar to data in <i>E</i>. <i>invadens</i> development, genes targeted by small RNAs were consistently silenced and did not change expression under tested stress conditions. Thus, the biological roles of the 27nt small RNA population in <i>Entamoeba</i> remain elusive. However, as the first characterization of the RNAi pathway in <i>E</i>. <i>invadens</i> these data serve as a useful resource for the study of <i>Entamoeba</i> development and open the door to the development of RNAi-based gene silencing tools in <i>E</i>. <i>invadens</i>.</p></div