Complete Genome Sequence of Fowl Adenovirus E from Gazipur, Bangladesh
Metagenomic libraries were constructed utilizing the iNextEra library preparation protocol. The DNA was tagmented using Illumina bead-linked transposomes (Illumina DNA library prep, Illumina, Inc., San Diego, CA, USA) and 2X TMP buffer in a total volume of 6 L, comprising 3 L of 2X TMP buffer, 0.5 L of bead-linked transposome, and 2.5 L of DNA, followed by incubation at 53C for 30 minutes. After tagmentation, a limited-cycle PCR step finalized the Illumina-compatible adapters with dual-indexes utilizing custom iNextEra primers for sequencing of pooled libraries. Each 51.5 L of PCR master mix comprised 40.25 L HyClone water (Cytiva Life Sciences, Marlborough, MA, USA), 9.5 L of Q5 Reaction Buffer (New England Biolabs, Ipswich, MA, USA), 1.25 L of 10mM dNTPs (Roche Diagnostic Corporation, Indianapolis, IN, USA), and 0.5 L of Q5 High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA). Subsequently, the master mix was incorporated into 6 L of the tagmentation mix. In conclusion, 2.5 L of 5 M iNextEra5 index primer and 2.5 L of 5 M iNextEra7 index primer were incorporated into the PCR mix, resulting in unique index combinations within a total PCR volume of 62.5 L. The limited-cycle PCR was performed using a T100 thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The protocol included an initial strand repair at 72C for 3 minutes, followed by denaturation at 98C for 3 minutes. This was succeeded by 12 cycles of 45 seconds at 98C, 30 seconds at 62C, and 2 minutes at 72C, concluding with a final extension at 72C for 1 minute. Following PCR, 5 L of the PCR product was analyzed on an agarose gel to confirm amplification of each sample, resulting in a smear within the target size range of 300-700 bp.