PNA-DNA clamp design
The probes in our study were designed based on the work of
Lundberg et
al. (2013) who developed PNA-DNA clamps to block mitochondrial (mPNA)
and chloroplast (pPNA) DNA, these clamps are known as universal clamps.
Recently,
Fitzpatricket al. (2018) reported that the universal pPNA showed a mismatch
in six plant lineages by means of an experimental and bioinformatic
analysis. Preliminary results from our study showed that the plant
contamination material in our bat samples belonged to one of these
lineages, namely Saguaro Columnar cacti (Cactaceae: Carnigea
gigantea ). Therefore, following the recommendations of Fitzpatricket al. (2018), we developed a special clamp for the bat samples
(Fig 2). This is not a trivial matter, since even a single base mismatch
between the chloroplast sequence and the clamp can increase levels of
plastid contamination in the sequencing output
(Fitzpatricket al. 2018). For the mockingbird, the universal clamps were used
to block both chloroplast and mitochondrial DNA. All clamps were ordered
from PNA Bio (Newbury Park, USA).