Phylogenetic placement of short reads
To further compare the primer biases at the sequence level, we used the
Rapid Axelerated Maximum Likelihood – Evolutionary Placement Algorithm
(RAxML-EPA; Berger, Krompass, & Stamatakis, 2011) approach to ascertain
the phylogenetic identities of the three abundant and important
phytoplanktonic groups in the Arctic, namely Chlorophyta,
Bacillariophyceae and Haptophyceae. To do this, phylogenetic reference
trees were generated for each taxonomic group using Randomized
Axelerated Maximum Likelihood (RAxML v.8; Stamatakis, 2014) based on
literature (Lovejoy et al., 2007; Liu, Aris-Brosou, Probert, & de
Vargas, 2010). HTS-generated OTUs were first searched against the NCBI
GenBank using BLASTn to identify the best hits. Most of the highly
similar sequences (> 98%, e-value 1e-10) were
selected and aligned in MUSCLE (Edgar, 2010), with the alignment
manually edited and trimmed in MEGA v.7.0 (Kumar, Stecher, & Tamura,
2016), and used to build robust clade-specific reference trees with
RAxML v.8 using the GTRGAMMA model, run 1000 times (Stamatakis, 2014).
Reference trees were generated separately for each taxonomic group.
Short OTUs were then aligned with the reference sequences and mapped
back onto the reference trees using the -n option in the EPA-RAxML v.8.2
(Berger et al., 2011). Trees generated from each primer pair were then
visualized in Dendroscope v.3.0 (Huson & Scornavacca, 2012) and
compared with each other.