Dna prep protocol

The library preparation was performed according to the Illumina DNA Prep protocol (Illumina, San Diego, CA, USA) [24 ]. The quantified DNA was first normalized and then added to the mixture of tagmentation reagents. The mixture was incubated on a thermocycler for 15 min at 55 °C. Furthermore, the post-tagmentation cleanup was performed by stopping the tagmentation reaction by adding the stop tagmentation buffer with further incubation at 37 °C for 15 min. The tagmented DNA was cleaned up by adding the tagmentation wash buffer to the mixture. Further, the amplification of the tagmented DNA was performed by the addition of the index (i7), index (i5), and oligos required for the cluster generation, as well as the addition of the enhanced PCR mix reagent. The amplified tagmented fragments of the library were purified on the magnetic stand using ethanol and Illumina purification beads (IPB). The purified library was further quantified on the Qubit 3.0 fluorometer (ThermoFisher Scientific, Waltham, MA, USA) and then normalized. The normalized library was then diluted and denatured to the final loading concentration to the v2 500 cycle Miseq reagent kit.

Libraries were built by using the Illumina DNA Prep protocol and Nextera DNA CD indexes (Illumina). Libraries were sequenced at the Perinatology Research Branch, using iSeq 100 reagents (Illumina) with the iSeq 100 system (Illumina) and an output of 2 × 150-bp paired-end reads.