Re-barcoding Rickettsia-containing BOLD DNA extracts
Aside from phylogenetic placement of these Rickettsia -containing
samples, attempts were made to extract an mtDNA barcode from these taxa
in order to identify the hosts of infected specimens. Previous
non-target amplification of Rickettsia through DNA barcoding of
arthropod DNA extracts had occurred in the bed bug Cimex
lectularius , with a recovery of the true barcode after using the primer
set C1‐J‐1718/HCO1490, which amplifies a shortened 455 bp sequence
within the COI locus. Subsequently, all samples were screened
using these primers or a further set of secondary COI primers
(LCOt_1490/ MLepR1 and LepF1/C_ANTMR1D) if the first failed to give an
adequate host barcode. All COI and Rickettsia multilocus
screening primer details, including references, are available in Table
S2.
Cycling conditions for COI PCRs were as follows: initial
denaturation at 95°C for 5 min, followed by 35 cycles of denaturation
(94°C, 30 sec), annealing (50°C, 60 sec), extension (72°C, 90 sec), and
a final extension at 72°C for 7 min. Rickettsia and host
amplicons identified by gel electrophoresis were subsequently purified
enzymatically (ExoSAP) and Sanger sequenced through both strands using a
BigDye® Terminator v3.1 kit (Thermo Scientific, Waltham, USA), and
capillary sequenced on a 3500 xL Genetic Analyser (Applied Biosystems,
Austin, USA). Forward and reverse reads were assessed in UGENE
(Okonechnikov,
Golosova, & Fursov, 2012) to create a consensus sequence by eye with a
cut-off phred (Q) score
(Ewing, Hillier,
Wendl, & Green, 1998) of 20. Primer regions were trimmed from barcodes
before being matched to Genbank and BOLD databases by BLAST based on
default parameters and an e-value threshold of <1e-85. Host
taxonomy was determined by a barcode-based assignment of the closest
BLAST hit, under the following criteria modified from Ramage et al.
(2017):
1) Species level designation for at least 98% sequence identity.
2) Genus level designation for at least 95% sequence identity.
3) Family level designation for at least 85% sequence identity.
Additionally, all sequences were required to be at least
>200 bp in length.