NGS-library preparation for whole-genome sequencing (WGS) starts with DNA fragmentation, and sonication is a physical approach used most often due to its simplicity and reproducibility. However, the commercially available Covaris instrument has a high price for both the device and consumables. Here we describe our in-house method of DNA shearing by sonication with small (100-600 µm) glass beads and an ultrasonic bath. The fragmentation conditions were optimized for the bacterial WGS with ∼550 bp fragment size (the ultrasonic bath water temperature 5–10 °C, glass beads 0.06 g, the fragmentation time 50 seconds), and for human DNA with ∼250 bp (fragmentation with the same parameters for 4 minutes). Fragmentation results were compared with the Covaris instrument for preparing several bacterial NGS libraries for Illumina NGS platforms by several characteristics. We obtained close mean fragment lengths (523–623 vs 480–646), similar mono- and dinucleotide specificity of shearing, and comparable indicators of read alignment and de novo assembly for both methods. Thus, the described method is a new fast, and effective DNA fragmentation approach that can be used in different WGS applications.

NTRK gene fusions are drivers of tumorigenesis events that specific Trk-inhibitors can target. Current knowledge of the downstream pathways activated has been previously limited to the pathways of regulator proteins phosphorylated directly by Trk receptors. Here, we aimed to detect genes whose expression is increased in response to the activation of these pathways. We identified and analyzed differentially expressed genes in thyroid cancer samples with NTRK1 or NTRK3 gene fusions, and without any NTRK fusions, versus normal thyroid gland tissues, using data from the Cancer Genome Atlas, the DESeq2 tool, and the Genome Enhancer and geneXplain platforms. Searching for the genes activated only in samples with an NTRK fusion as opposed to those without NTRK fusions, we identified 29 genes involved in nervous system development, including AUTS2, DTNA, ERBB4, FLRT2, FLRT3, RPH3A, and SCN4A. We found that genes regulating the expression of the upregulated genes (i.e., upstream regulators) were enriched in the “signaling by ERBB4” pathway. ERBB4 was also one of three genes encoding master regulators whose expression was increased only in samples with an NTRK fusion. Moreover, the algorithm searching for positive feedback loops for gene promoters and transcription factors (a so-called “walking pathways” algorithm) identified the ErbB4 protein as the key master regulator. ERBB4 upregulation (p-value = 0.004) was confirmed in an independent sample of ETV6-NTRK3-positive FFPE specimens. Thus, ErbB4 is the potential key regulator of the pathways activated by NTRK gene fusions in thyroid cancer. These results are preliminary and require additional biochemical validation.