Sequencing of the Exon 2 of the MHC Class II DQB Locus in the Caspian seal
Total DNA from samples of fresh collected muscle tissue and blood was extracted using the Magna DNA Prep kit (Isogen, Russia) on the KingFisher Flex 96 magnetic particle processor (Thermo Fisher, Finland). DNA from hair and postmortem material samples was extracted using the QIAamp DNA Investigator kit (Qiagen, Germany). Further library preparation and sequencing was performed in the commercial laboratory of LLC Evrogen Lab (Moscow, Russia). Individual libraries were prepared following the "16S Metagenomic Sequencing Library Preparation" guide (Part # 15044223 Rev. B; Illumina). Amplicons were obtained using primers based on DQB_F: 5- GATTTCGTGTACCAGTTTAAGGGC -3 and DQB_R: 5- CCACCTCGTAGTTGTGTCTGCA -3 sequences given in Sonsthagen et al., 2014, DOI: 10.1007/s00300-014-1450-9). After obtaining amplicons, the libraries were purified and equimolarly pooled using the SequalPrep Normalization Plate Kit (ThermoFisher, Cat # A10510-01). Quality control of the resulting library pools was performed using the Fragment Analyzer system, and quantitative analysis was conducted through qPCR. The library pool was sequenced on the Illumina MiSeq using MiSeq Reagent Kit v2 Nano reagents: read length of 250 bp from both ends of the fragments, 500 cycles. A PhiX phage library was used for sequencing parameter control.