Characteristics of reads aligned to the HIV-1_JR-FL genome identified by AGO-2 PAR-CLIP in HIV-1_JR-FL infected (n = 2) and non-infected (n = 2) samples.

<p>^aThe location of each cluster mapping to the HIV-1_JR-FL genome is specified by its start and end position according to the HIV-1_HXB2 reference genome (GenBank accession number K03455)</p><p>^bThe coverage shows the total number of reads aligned to loci</p><p>^cT-to C counts are the numbers of observed T-to C mutations in the aligned reads</p><p>^dAnalysis of protein-binding (indicated with “+”) by PARAlyzer [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132127#pone.0132127.ref044" target="_blank">44</a>] analysis pipeline</p><p>Characteristics of reads aligned to the HIV-1_JR-FL genome identified by AGO-2 PAR-CLIP in HIV-1_JR-FL infected (n = 2) and non-infected (n = 2) samples.</p

High-throughput sequencing of small RNA libraries of HIV-1_JR-FL infected (n = 4) and non-infected (n = 4) monocyte-derived macrophages.

<p>^aTotal reads represent reads after size selection (≥13 nts) and removal of adaptor-adaptor sequences</p><p>^bReads were competitively aligned to the human and the HIV-1_JR-FL genome. Read number aligned to either both genomes or HIV-1_JR-FL only are shown (Reads aligned) in addition to % of all reads (Reads aligned in %, Reads aligned to HIV-1_JR-FL in %).</p><p>High-throughput sequencing of small RNA libraries of HIV-1_JR-FL infected (n = 4) and non-infected (n = 4) monocyte-derived macrophages.</p

Characteristics of small RNAs derived from small RNA sequencing.

<p>Small RNA species detected in HIV-1_JR-FL infected MDMs (n = 4) (<b>A</b>), and non-infected MDMs (n = 4) (<b>B</b>). (<b>C</b>) Read length distribution of HIV-1 sncRNAs (dark grey) and total small RNA (light grey). (<b>D</b>) Small RNA sequencing reads (n = 4) aligned to the HIV-1_JR-FL genome. Transcripts aligned in antisense orientation are shown in red, predominantly representing tRNA^Lys. Upper panel shows a diagram of the HIV-1 genome organization.</p

Cellular micro RNAs as detected by Ago2 PAR-CLIP and small RNA sequencing.

<p><b>(A)</b> Percentage of the cellular miRNAs from small RNA sequencing that was also found in Ago2 PAR-CLIP as a function of minimum expression threshold. The x-axis shows the total abundance of miRNAs in small RNA sequencing data derived from pooled HIV-1_JR-FL infected (n = 4) and HIV-1 non-infected (n = 4) samples from the same donors. Red lines show the read count corresponding to specific fractions of total miRNA pool <b>(B)</b> Expression of cellular miRNAs in Ago2 PAR-CLIP and small RNA sequencing data is well correlated for 160 miRNAs found by both methods (<i>R</i> = 0.55, <i>p</i><10^‒13). The dashed lines show bootstrap-derived 95% confidence intervals for the linear fit (red line). <b>(C)</b> Expected PAR-CLIP read counts of virus-derived sncRNAs associated with Ago2. Adjusted to small RNA sequencing data derived from pooled HIV-1_JR-FL infected (n = 2) samples matching the PAR-CLIP donors. The black line (5 reads aligned) depicts the detection limit of the PAR-CLIP assay with majority of the loci on the sense genome are expected to surpass the detection limit. Upper and lower panels correspond to the reads on the sense and anti-sense strand of HIV-1_JR-FL genome respectively.</p

Frequencies of true and false haplotypes.

a<p>see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074249#pone-0074249-t002" target="_blank">table 2</a>.</p><p>n.p. qPCR was not performed.</p><p>n.a. not applicable.</p

Substitution and insertion/deletion rates per base of different procedures for amplicon generation.

*<p>each sample was done in duplicate, the sample name a and b helps to distinguish the duplicates from each other.</p><p>NGS, next-generation sequencing; RT, reverse transcription.</p

Substitution rates of each virus strain used to generate the 5-virus-mix.

<p>Each of the HIV-1 stocks was pyrosequenced separately to control for the purity of each virus strain. The y-axis shows the substitution rate per base according to the reference within the analyzed 271 bp long fragment (amino acids 10–99 of the HIV-1 protease, nt 2279–2549 based on HIV-1_HXB2). The x-axis shows the positions on the sequence. The orange bars indicate differences in the nucleotide sequences of the five virus strains.</p

Substitution and insertion/deletion rates and their sources using 454 pyrosequencing.

<p>A) The molecular full-length HIV-1 clone pYK-JRCSF was used to generate three different samples for the determination of error rates and error sources during the different steps of sample preparation. The blue (left) pathway indicates the procedure NGS, i.e., no amplification step was performed before emulsion PCR and pyrosequencing. The green (right) pathway shows the procedure PCR-NGS, i.e., the target was amplified once prior to 454 emulsion PCR/pyrosequencing. The orange (middle) pathway depicts the commonly used procedure RT-2PCR-NGS to reverse transcribe, amplify and sequence HIV-1 RNA genomes reflecting the errors that will occur using patients’ plasma samples to analyze HIV-1 haplotypes. Detailed description of each step is given in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074249#s4" target="_blank">materials and methods</a> section. B) Error rates per positions are shown for forward reads (left) and reverse reads (right). For each duplicate, one example is shown (always sample a as presented <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074249#pone-0074249-t001" target="_blank">table 1</a>).</p

Major <i>in vitro</i> recombinant haplotypes assigned by ShoRAH.

<p>Haplotypes were aligned to the five reference strains and characterized. The top part shows the five virus strains (true haplotypes) of the 5-virus-mix and the bars indicate the specific mutation for each strain distinguishing it from the other four virus strains. The corresponding nucleotides and positions are indicated. HIV-1_HXB2 has one unique mutation at position 84 (reference numbering 2362) that is indicated in grey. The mutations for HIV-1_NL4-3 are marked in blue, in HIV-1_JR-CSF in green, in HIV-1_89.6 in red, and in HIV-1_YU2 in orange. Dark colours indicate unique mutations, light colours indicate differences to other strains but not unique for the respective strain. The bottom part shows all recombinant haplotypes found at 1% and higher frequencies. Triangles indicate positions were a specific nucleotide is expected according to the corresponding strain, but is missing. The Nucleotide positions in the sequences are indicated.</p

Adverse events and laboratory abnormalities during silibinin treatment.

<p>¹ All adverse events were of mild or moderate intensity and resolved spontaneously</p><p>² The Increase in bilirubin was clinically not significant and resolved spontaneously</p><p>Adverse events and laboratory abnormalities during silibinin treatment.</p