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Targeted locus amplification (TLA) allows for the detection of all genetic variation (including structural variation) in a genomic region of interest. As TLA is based on proximity ligation, variants can be linked to each other, thereby enabling allelic phasing and the generation of haplotypes. This allows for the study of genetic variants in an allele-specific manner. Here, we provide a step-by-step protocol for TLA sample preparation and a complete bioinformatics pipeline for the allelic phasing of TLA data. Additionally, to illustrate the protocol, we show the ability of TLA to re-sequence and haplotype the complete cystic fibrosis transmembrane (CFTR) gene (> 200 kb in size) from patient-derived intestinal organoids.

Targeted locus amplification (TLA) allows for the detection of all genetic variation (including structural variation) in a genomic region of interest. As TLA is based on proximity ligation, variants can be linked to each other, thereby enabling allelic phasing and the generation of haplotypes. This allows for the study of genetic variants in an allele-specific manner. Here, we provide a step-by-step protocol for TLA sample preparation and a complete bioinformatics pipeline for the allelic phasing of TLA data. Additionally, to illustrate the protocol, we show the ability of TLA to re-sequence and haplotype the complete cystic fibrosis transmembrane (CFTR) gene (gt; 200 kb in size) from patient-derived intestinal organoids.

Chromosomal translocations with immunoglobin (IG) loci are the classic drivers in a large subset of B-cell lymphomas. Detection of these translocations is important for confirmation of diagnosis and for prognosis and therapy decisions. Currently, molecular diagnosis of translocations in lymphomas is not addressed well by next-generation sequencing (NGS). The standard method for detection of translocations is fluorescence in situ hybridization (FISH), which is labor intensive and can be difficult to interpret. There is a need for a robust technology that can be standardized. Targeted Locus Capture (TLC) selectively enriches and sequences entire genes based on the crosslinking of physically proximal sequences, and thereby enables complete sequencing of genes of interest, including detection of large structural variants. Because the technology is based on the crosslinking and fragmenting of DNA, it has particular advantages in the analysis of formalin-fixed, paraffin-embedded (FFPE) samples in which DNA is inherently crosslinked and fragmented. In order to validate the FFPE-TLC technology as a novel approach for translocation detection in lymphoma samples, we have developed a panel assay containing genes with frequent translocations (MYC, BCL2, BCL6, IG loci). With this assay we have analyzed >140 lymphoma and control FFPE samples of variable input amounts and qualities that had previously been analyzed with FISH, and a subset also with standard targeted NGS. Good concordance with FISH results was observed for both translocation-positive and -negative samples. In 10 cases for which FFPE-TLC analysis resulted in a different finding than FISH, discordance could be explained by higher sensitivity of FFPE-TLC or by inconclusive FISH results. In a specific case, FFPE-TLC detected a small-distance rearrangement on chromosome 3 that caused a BCL6 fusion but led to insufficient and therefore undetectable break-apart with FISH. Secondly, the FFPE-TLC approach was tested on a set of 19 B-cell lymphoma FFPE samples that had previously been analyzed using standard targeted NGS and FISH and was enriched for discordant results between these methods. FFPE-TLC-based NGS enables more robust translocation calling as the detection relies on broad sequencing coverage across the translocation partner rather than on breakpoint sequences only. In 3 cases, FFPE-TLC could proof false negative calls in standard targeted NGS due to breakpoints located in regions difficult to capture or to sequence. In 1 case, standard targeted NGS had made a false positive call on a breakpoint sequence that was shown to be caused by a small insertion rather than a genuine translocation. This study shows that FFPE-TLC promises to be a robust alternative for FISH analysis and standard targeted NGS procedures in lymphoma diagnostics and in other cancers with frequent structural variants. The FFPE-TLC approach enables a single, DNA-based NGS test detecting both small mutations and translocations. Citation Format: Amin Allahyar, Mark Pieterse, Joost Swennenhuis, Tjitske Los-de Vries, Mehmet Yilmaz, Roos Leguit, Ruud Meijers, Nathalie Hijmering, Daphne de Jong, Bauke Ylstra, Robert van der Geize, Joost Vermaat, Arjen Cleven, Tom van Wezel, Arjan Diepstra, Leon van Kempen, Karima Hajo, Harma Feitsma, Marieke Simonis, Max van Min, Erik Splinter, Wouter de Laat. Robust detection of translocations in lymphoma FFPE samples using Targeted Locus Capture-based sequencing [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-45.

We present TaxPhlAn, a new method and bioinformatics pipeline for design and analysis of single-locus sequence typing (SLST) markers to type and profile bacteria beyond the species-level in a complex microbial community background. TaxPhlAn can be applied to any group of phylogenetically-related bacteria, provided reference genomes are available. As TaxPhlAn requires the SLST targets identified to fit the phylogenetic pattern as determined through comprehensive evolutionary reconstruction of input genomes, TaxPhlAn allows for the identification and phylogenetic inference of new biodiversity. Here, we present a clinically relevant case study of high-resolution Staphylococcus profiling on skin of atopic dermatitis (AD) patients. We demonstrate that SLST enables profiling of cutaneous Staphylococcus members at (sub)species level and provides higher resolution than current 16S-based techniques. With the higher discriminative ability provided by our approach, we further show that the presence of Staphylococcus capitis on the skin together with Staphylococcus aureus associates with AD disease.

Background Structural variants in DNA such as translocations are increasingly found as key oncogenic events in tumors, and detection is clinically relevant for therapy choice in many conditions. Despite developments in targeted and whole genome gene sequencing, the robust detection of structural variants remains a challenge, especially in FFPE specimens. Methods Targeted proximity ligation assays selectively amplify and sequence entire genes based on the crosslinking of physically proximal sequences, and thereby enable complete sequencing of genes of interest, including single nucleotide and structural variants. Because these assays are based on the crosslinking and fragmenting of DNA, they have particular advantages in the analysis of FFPE samples, in which DNA is inherently crosslinked and fragmented. Results We will show that we have successfully developed targeted proximity ligation assays on representative clinical FFPE samples with considerably fragmented DNA. By making use of the DNA crosslinks in these samples, we can generate very broad sequence information over 100’s of kb’s, where other targeted NGS technologies are limited by the fragment size. We will show that we can detect NTRK fusions at DNA level, including determination of the fusion partner and the exact translocation breakpoint sequence. The latter can be used as a very sensitive and cancer-specific marker for minimal residual disease (MRD) testing and ctDNA monitoring with PCR. Conclusions Our newly developed targeted proximity ligation assays enable robust detection of structural variation in FFPE samples and the development of personalized MRD/ctDNA tests. Legal entity responsible for the study Cergentis. Funding Cergentis via Horizon2020 SME funding. Disclosure H. Feitsma: Full / Part-time employment: Cergentis. M. Yilmaz: Full / Part-time employment: Cergentis. J. Swennenhuis: Full / Part-time employment: Cergentis. A. Rakszewska: Full / Part-time employment: Cergentis. K. Hajo: Full / Part-time employment: Cergentis. E. Splinter: Full / Part-time employment: Cergentis. M. Simonis: Full / Part-time employment: Cergentis. M. Van Min: Shareholder / Stockholder / Stock options, Full / Part-time employment: Cergentis. All other authors have declared no conflicts of interest.

Lactobacillus delbrueckii subsp . bulgaricus LBB.B5 originates from homemade Bulgarian yogurt and was selected for its ability to form a strong association with Streptococcus thermophilus . The genome sequence will facilitate elucidating the genetic background behind the contribution of LBB.B5 to the taste and aroma of yogurt and its exceptional protocooperation with S. thermophilus .