Truseq nano kit

The DNA labeled with ssARP was bound to the Dynabeads MyOne Streptavidin C1 (Invitrogen), the beads were washed with the manufacturer recommended 2× DNA binding and wash buffer (B&W buffer) and were separated from the solution on a magnetic stand (DynaMag, Invitrogen). The supernatant containing the unbound DNA was removed, the beads were washed with 1× B&W buffer, and resuspended in 1× TE. The bound DNA was released from beads by incubating with 100 mM dithiothreitol for 10 min at 37°C. Beads were placed on the magnetic stand, and the supernatant which contained the eluted DNA was collected. DNA was concentrated using ethanol precipitation.
The pulled-down DNA was used for DNA library preparation using Illumina TruSeq nano kit (Illumina). All the libraries were pooled in equimolar quantities for multiplexed sequencing and sequenced on Illumina MiSeq platform (Michigan State University). The sequencing was performed in a 2× 150 bp paired-end format using a MiSeq v2 300 cycle reagent cartridge. Base calling was done by Illumina Real Time Analysis (RTA) v1.18.54 and output of RTA was demultiplexed and converted to FastQ format using Illumina Bcl2fastq v2.19.1. The list of fastq files and their accession numbers are provided in Supplementary Table S3.

The 16 S rRNA amplification and high-throughput sequencing was performed by Molecular Research LP (MR DNA; Shallowater, TX, USA) following the protocol described by Vargas et al. 2023 [70 (link)]. Briefly, each sample of DNA was diluted to 25 ng/μL. The DNA of three fish per pond were mixed in equal volumes to form a pool. For each condition, two pools were generated. Each DNA pool was used as a template to amplify the V4 variable region using primers 515-F and 806-R [71 (link)] with a HotStarTaq Plus Master Mix Kit (Qiagen, Hilden, Germany). The PCR conditions were set up to 94 °C (3 min), 30 cycles of 94 °C (30 s), 53 °C (40 s), and 72 °C (60 s), with a final elongation to 72 °C (5 min). PCR products were analyzed in a 2% agarose gel, purified using Ampure XP beads. Illumina DNA library using purified PCR products was prepared with a TruSeq Nano kit. MiSeq reagent kit v3 on the Illumina MiSeq platform (2 × 300-bp paired ends [PE]) was used for high-throughput sequencing of amplicons following the manufacturer’s guidelines.