Rsii system

Concurrently, individual carbapenem-resistant K. pneumoniae colonies (n = 14) were picked at random from patient A’s primary sample plated on Brilliance CRE selective media (Thermo Fisher Scientific, Waltham, MA, USA). Genomic DNA from each colony was extracted using a JANUS automated workstation with a Chemagic viral DNA/RNA kit (PerkinElmer). Libraries were prepared as described above and sequenced on a NextSeq 500 system with 150-cycle paired-end chemistry as described in the manufacturer’s protocols (Illumina Inc., San Diego, CA, USA).
Representative KPC-producing K. pneumoniae isolates from previously defined local transmission networks were used as comparative genomes for this study (38 (link)). Three isolates were selected, all from different local transmission networks involving patients colonized or infected with KPC-producing K. pneumoniae, with the networks defined through combined genomic and epidemiological inference. DNA extraction, size selection, and sequencing on a Pacific Biosciences RS II system (Pacific Biosciences, Menlo Park, CA, USA) were performed as previously described (39 (link)), with additional Illumina sequencing data used for polishing to produce high-quality closed genomes.
Sequencing data from this study are available through the European Nucleotide Archive (BioProject PRJEB23689; see reference 38 (link) for K. pneumoniae genomes).

Genomic DNA from two strains of Francisella, FSC996 and FSC100616 (link), were extracted using a previously described phenol/chloroform method17 (link). The isolated DNA was used for sequencing with a MinION system (Oxford Nanopore Technologies Ltd [ONT], Oxford, UK) in-house. DNA preparations were also sent for sequencing by a Pacific Biosciences RS II system (10 kb library, P4-C2 chemistry, 120 min data collection) at the Uppsala Genome Center (Uppsala, Sweden) and for Illumina HiSeq 2000 sequencing (100 bp, paired end) at SNP&SEQ (Uppsala, Sweden). All sequencing operations were performed using the reagents and protocols recommended by the manufacturers.

Aliquots of 2x10⁶ B cells were processed according to the ARTISAN PCR protocol for unbiased amplification of BCR repertoires, which has very low amplification and sequencing error rates of 0.126 × 10⁻³ per bp (45 (link)). Full-length IgM and IgG VDJ were amplified from all twelve donors, while IgA and IgE VDJ. VJ-kappa and VJ-lambda were amplified from six donors each (46 (link)). Libraries were barcoded, pooled and amplified as single molecules in rolling circles on a total of fourteen SMRT cells on the RSII system (Pacific Biosciences, Menlo Park, CA, USA). Output sequence files were filtered with SMRT portal software for a minimum of eight sequencing passes. All sequences were annotated by IMGT HighV-QUEST (47 (link)).
Additional datasets of healthy donors, as well as UNG-deficient and MSH2- and MSH6-deficient patients were obtained from publicly available sources (48 (link), 49 (link)) (Supplementary Table S1). Its amplification and sequencing methodology was previously validated and shown to be reliable for the identification VDJ of naïve and memory B cells alike (50 (link)).

Postcapture libraries (2- to 5-µg samples) were sent to the Centre for Genomic Research at the University of Liverpool, where SMRTbell adapters were added and sequencing was performed using a PacBio RS II System with P6/C4 chemistry. In most cases, libraries were sequenced using a single SMRT cell. However, in a few cases two SMRT cells were used.

The repeat size was determined by (TP-)PCR for all samples. The structure of the CGG repeat in the FMR1 gene was then determined for all intermediate and premutation carriers by sequencing according to the previously published method (Ardui et al., 2017 (link)). In brief, the FMR1 CGG repeat was amplified by PCR during which a barcode was incorporated. After pooling different amplicons together, long-read single-molecule sequencing was performed on a PacBio RSII system. This generates long reads which span each PCR molecule multiple times thereby generating a highly accurate consensus sequence. This technology is “single-molecule” which allows the unambiguous separation of the CGG repeats derived from the two different X-chromosomes. Hence, after sequencing, the complete repeat structure could be reconstructed for both X-chromosomes revealing the repeat size and the AGG interruption pattern. The sizes determined by PCR control runs and single-molecule sequencing matched perfectly.