Amplicon Sequence of Saxicola_maurus_SRIB0391 (Ring Number JC63451) [Raw reads]
Feather-derived DNA was extracted from the rectrices (tail feathers). DNA extractions followed the QIAGEN DNeasy Blood & Tissue Kit protocol with minor modifications, using 50 L AE buffer for each of two elutions. DNA concentration was quantified with a Qubit 4 fluorometer (Thermo Fisher Scientific). The mitochondrial Cytochrome Oxidase I (CO1) barcode region (658 bp) was amplified using primers FF2d (Ivanova et al. 2007) and CO1R (Dove et al. 2008), selected for passerine specificity and high combined coverage across birds (de Melo et al. 2021). PCR reactions were conducted using Thermo Fisher Scientific Hot Start DNA Polymerase under standard conditions, with cycling parameters consisting of an initial denaturation at 94 C for 3 min, followed by 40 cycles of 94 C for 15 s, 50 C for 30 s, and 72 C for 40 s, and a final extension at 72 C for 5 min. Amplicon concentrations were verified using Qubit fluorometry. Indexed PCR products were pooled equimolarly, targeting 120 ng of DNA per sample. One extraction with insufficient yield contributed all available amplicons. The pooled library was purified using AMPure XP beads (1:1.2 bead-to-sample ratio) and prepared for sequencing with the Oxford Nanopore Technologies (ONT) Ligation Sequencing Kit (SQK-LSK114), using reagents from the NEBNext Companion Module. Libraries were sequenced on an ONT MinION Mk1C using a Flongle (FLO-FLG114HD), with high-accuracy basecalling enabled, read splitting activated, a minimum read length of 200 bp, and sequencing run until pore exhaustion.