454 gs junior instrument

Mice were immunised orally with sheep red blood cells for 2 weeks and intraperitoneally with 10 μg of LPS for 3 days. Single-cell suspensions from Peyer's patches were labelled with B220-APC- (Biolegend, ref:103212), GL7-FITC- (Beckton Dikinson, ref: 561530) and Fas-PE- (Beckton Dikinson, ref: 554258) conjugated antibodies. Purification of B220⁺GL7⁺Fas⁺ cells was realized on a FACS ARIA III (BD). Genomic DNA was extracted, and a region corresponding to a sequence of 517 bp downstream of the J_H4 segment was amplified by PCR. As a control, Igκ light-chain VJ-rearranged fragments were also amplified. Primers (detailed in the Supplementary Table 1) were coupled to 454 Sequencing adaptor sequences and PCR was performed using the program previously reported8 (link). According to the manufacturer, the resulting purified amplicons were prepared for sequencing with a GS Junior Titanium emPCR Kit (Lib-A; Roche), and the library of DNA fragments was sequenced on a 454 GS Junior instrument (Roche). Obtained sequences were aligned to the reference sequence using BWA aligner38 , and SAMtools software was used to obtain BAM files39 (link). Redundant sequences were excluded, and wig files were generated using IGV Tools40 (link) and manually analysed to determine mutation frequencies for each nucleotide in the sequence.

Viral genetic diversity, together with the presence of stop codons/hypermutations, was analyzed based on an amplicon from the RT region of the pol gene (20 (link)). Amplification was carried out as previously described by the ANRS French resistance group (HXB2 coordinates of the amplicon, 2609 to 3292) (44 ), with an adapted nested PCR including the multiplex identifier (MID) of the 454 GS Junior instrument (Roche Diagnostics). Deep sequencing of the RT amplicon was performed using GS Junior sequencing XL+ kits, which provided reads up to 800 bp long, following the manufacturer’s instructions. The 8E5/LAV cell line was included as a control.

Samples were processed for viral metagenomics as described previously [9 (link),49 (link)]. In brief, samples were depleted from host nucleic acids and filtered through a 0.45 μM filter. Subsequently, RNA and DNA were extracted using the Nucleospin RNA XS kit (Macherey-Nagel) and the High Pure viral nucleic acids kit (Roche). First and second strand synthesis and random PCR amplification were performed. PCR products were purified and processed for next-generation sequencing with a 454 GS Junior Instrument (Roche). Obtained reads were assembled using de novo assembly in CLC Genomics Workbench 5 (CLC Bio) and contigs and individual reads were analyzed by BLASTN and BLASTX respectively. Cut off E-values for significant virus hits for BLASTN and BLASTX were respectively 1.0 × 10⁻³ and 1.0 × 10⁻¹⁰. Based on the taxonomic origin of the best-hit sequence, classification of the sequences was performed in MEGAN 4.70.4 [53 (link)]. Obtained reads were deposited at the European Nucleotide Archive under archive number PRJEB4910.