Additional file 5: of Small RNA profiling of low biomass samples: identification and removal of contaminants

Figure S4. Relative abundance of potential exogenous sRNAs in datasets derived from a plasma sample of one healthy individual. Detected levels of the 21 potential exogenous sRNA sequences in preparations using 45 to 1115 μL human plasma and regular or ultra-clean RNeasy spin columns and in controls without plasma, including no library, mock extractions and water controls (n = 33). cpm counts per million. Error bars indicate one standard deviation; data points are available in Additional file 2: Table S11. (PDF 11 kb

Additional file 3: of Small RNA profiling of low biomass samples: identification and removal of contaminants

Figure S2. Detection of contaminants in published datasets. Heatmap showing the relative abundances of the confirmed contaminant sequences in published sRNA sequencing data of low-biomass samples. Only samples for which any of the confirmed contaminants were detected are shown. Extraction methods: Q regular QIAGEN miRNeasy; T TRIZOL. rpm reads per million. (PDF 106 kb

Additional file 2: of Small RNA profiling of low biomass samples: identification and removal of contaminants

Table S1. List of the generated datasets with public accession numbers. Table S2. Analysed published datasets with references and public accession numbers. Table S3. Potential exogenous sRNA sequences detected in human plasma after removal of contaminants. Table S4. List of the prokaryotic species whose reference genomes were used in the initial analysis. Table S5. List of the eukaryotic species whose reference genomes and/or cDNA collections were used in the initial analysis. Table S6. List of the viruses whose reference genomes were used in the initial analysis. Table S7. Data points for Fig. 2a. Table S8. Data points for Fig. 2b. Table S9. Data points for Fig. 2c. Table S10. Data points for Fig. 2d. Table S11. Data points for Fig. 4a. Table S12. Data points for Fig. 5b. (XLSX 228 kb

Additional file 4: of Small RNA profiling of low biomass samples: identification and removal of contaminants

Figure S3. Detection of contaminants in eluates of regular and ultra-clean RNeasy columns. Two batches of regular miRNeasy columns and four batches of ultra-clean RNeasy columns were compared. Results are based on sRNA sequencing data of mock extracts, normalised to the detected levels of spike-in synthetic RNAs. The different shadings represent reads mapping to the human genome with 2, 1, or 0 mismatches and the different column batches are coloured in the same colours as in main Fig. 3, as indicated in the legends. (PDF 16 kb

Additional file1: of Small RNA profiling of low biomass samples: identification and removal of contaminants

Figure S1. Scheme summarising the different control experiments, the titration experiments and their outcomes. a) Tracing non-human sRNA sequences to contaminants on spin columns by variation of different steps in the isolation protocol and analysis by qPCR assays. Modifications to the steps named at the top are listed below the workflow and the outcomes are summarised at the right hand side. b) Workflow of the titration experiment to determine a minimal safe input volume for all contaminant sequences. UCP column ultra-clean column. (PDF 86 kb

The c.-547C>T mutation in the <i>AR</i>-5′UTR reduces translation and AR activity <i>in vitro</i>.

<p>HEK293 cells were transfected with either empty vector, <i>AR</i>5′UTRwt-<i>GFP</i> or <i>AR</i>5′UTRmut-<i>GFP</i>. After 72h of transfection RNA and protein was isolated. A) Q-RT-PCR analysis of GFP mRNA. There is no significant difference between <i>GFP</i> mRNA levels of the <i>AR</i>5′UTRwt-<i>GFP</i> and the <i>AR</i>5′UTRmut-<i>GFP</i> construct (p = 0.57). <i>GFP</i> mRNA levels were normalized to the neomycin resistance (neo) expression of the vector. Experiments were performed in triplicate. B) GFP protein analysis. Cells transfected with the <i>AR</i>5′UTRmut-<i>GFP</i> construct show a reduced expression of the GFP protein compared to the wt-construct. GAPDH measurement served as loading control. The experiment was done in triplicate. One representative blot is displayed. C) FACS analysis of <i>AR</i>5′UTR-<i>GFP</i> transfected cells. FACS analysis was performed equally 72h after transfection. Cells transfected with <i>AR</i>5′UTRmut-<i>GFP</i> show less fluorescent intensity. D) Transcriptional activity of <i>AR</i>5′UTRwt-<i>AR and AR</i>5′UTRmut-<i>AR</i>. HEK293 cells were transfected with either empty vector, <i>AR</i>5′UTRwt-<i>AR</i> or <i>AR</i>5′UTRmut-<i>AR</i>. After 48h of transfection luciferase activity was measured. AR induced luciferase expression is significantly lower in <i>AR</i>5′UTRmut-<i>AR</i> transfected cells in respect to <i>AR</i>5′UTRwt-<i>AR</i> transfected cells (p***<0.001).</p

Additional data on index patient 1.

<p>Additional data on index patient 1.</p

Next generation sequencing of the AR in the two index patients.

<p>A) Schematic representation of the <i>AR</i>-5′UTRs and CDS. The position of the c.-547C>T mutation and the uORF are indicated. B) A Haloplex library spanning the coding region, introns, UTRs and up and downstream sequences of the AR genomic locus was prepared from DNA of the index patients′ GF and sequenced on a MiSeq benchtop sequencer. Analysis for single nucleotide polymorphisms (SNP) and small insertion deletions (indels) was performed by the SureCall software (Agilent). Indicated is the mutation found in the 5′UTR of the <i>AR</i>. A frequency of 1 corresponds to 100% of the reads. The depth indicates the number of reads covering the indicated genomic position. C) Sanger sequencing of a male control and the index patients 1 and 2. The sequences are visualized as reverse complement strand using the Chromas Lite software and show the c.-547C>T mutation in both index patients but not in the male control. D) Sanger sequencing of blood derived DNA from both index patients as well as from the mothers of the index patients. The sequences are visualized as reverse complement strand using the Chromas Lite software and show the c.-547C>T mutation in both index patients and in the patients′ mothers in a heterozygous constellation.</p

Additional data on index patient 2.

<p>Additional data on index patient 2.</p

Functional and expression analysis of the AR <i>in vivo</i> and <i>in vitro</i>.

<p>A) <i>AR</i> mRNA accumulation in GF. Total RNA was extracted from GF of a male control, a CAIS patient with a documented frameshift mutation in the <i>AR</i> gene and index patient 1. Means and standard deviations of three independent experiments are shown. B) DHT-dependent AR protein expression in GF. Whole cell protein lysates were extracted from GF of the male control, the index patient and the CAIS patient with a documented frameshift mutation and treated with 10nM DHT or ethanol, respectively. From all samples, 35 μg of protein were loaded, additionally 10 μg were loaded for the control sample to avoid overexposition. Immunoblot analysis shows a markedly reduced amount of the 112 kD full-length AR protein in the index patient and an increased detection of a shorter 75kD fragment. DHT treatment stabilizes full-length 112kD AR protein while the shorter 75kD fragment remains of similar intensity as compared to untreated GF. The corresponding bands are indicated by an arrow. An unspecific band is denoted by a star. GF from the CAIS patient served as negative control. Actin measurement served as loading control. C) DHT-dependent ectopic AR protein expression in PC3 cells. Cells were either treated with 10nM DHT or ethanol. 5′UTRmut-<i>AR</i> transfected cells show reduced AR protein expression compared to 5′UTRwt-<i>AR</i> transfected cells. AR-121 transfected cells express an AR-fragment of lower molecular weight. Actin measurement served as loading control. D) DHT-dependent activation of the AR target gene <i>APOD</i> is compromised in the index patient. AR activity was measured through the activation of the endogenous AR target gene <i>APOD</i>. This revealed a mean 3.4 fold activation of <i>APOD</i> in response to DHT stimulation in three male foreskin control derived cell lines. GF of the index patient, like GF of the CAIS patient with known mutation show a highly significant loss of <i>APOD</i> induction as compared to the male controls (p***<0.001). Means and standard deviations of three independent experiments are shown. p-values are calculated by a t-test. E) DHT-dependent activation of the AR target gene <i>PPAP2B</i> is compromised in the index patient. AR activity was measured through the activation of the endogenous AR target gene <i>PPAP2B</i>. This revealed a mean 1.54 fold activation of <i>PPAP2B</i> in response to DHT stimulation in three male foreskin control derived cell lines. GF of the index patient, like GF of the CAIS patient with known mutation show a significant loss of <i>PPAP2B</i> induction as compared to the male controls (p**<0.01). Means and standard deviations of at least three independent experiments are shown. p-values are calculated by a t-test.</p