MODY in young-Bangladeshi
Genomic DNA (gDNA) was extracted using QIAamp DNA Mini Kit (cat. nos. 51304). Library preparation, enrichment, and amplification were performed using custom capture kit from Medgenome, India. In the laboratory of DNA Solutions Ltd., Dhaka. gDNA was fragmented using enzymatic fragmentation, followed by end repair and A-tailing to prepare the DNA for adapter ligation. The fragmented, A-tailed DNA was then ligated to a Twist universal adapter, ensuring compatibility for subsequent amplification. Post-ligation, the DNA was purified to remove unligated fragments and other contaminants. The purified libraries underwent PCR amplification using unique dual index (UDI) primers to generate indexed libraries, which were then purified again to remove residual primers and dNTPs. Target enrichment was achieved through hybridization with capture probes specific to the MODY genes, followed by binding to streptavidin beads. The captured target sequences were washed and subjected to post-capture PCR amplification to enrich for the target regions. The final libraries were validated for quality and size, with an average library size between 370-430 bp and DNA concentration 2 ng/L. For multiplexing, DNA from different samples was pooled using unique indexes for each, enabling sequencing of multiple samples in a single run. Finally, the MODY genes were sequenced in the laboratory of the National Institute of Biotechnology, Savar, Dhaka using the Illumina NovaSeq 6000 (Illumina, CA).