Sequencher v4

Peripheral blood was obtained from the patient and their parents after informed consent. Genomic DNA and total RNA were extracted from the blood by standard methods. Genomic fragments containing the variants identified by WES were amplified by PCR and sequenced for both strands. Total RNAs (1 μg) were used to synthesize cDNA with a PrimeScript RT reagent Kit (Takara Bio). The electrophoretic bands of the PCR products were cut from the gel, purified, and sequenced. The PCR primer sets are shown in Supplementary Table 3. A 3730 DNA analyzer (Life Technologies) was used for the Sanger sequencing. Sequencher V.4.7 (Gene Codes) and Genetyx Ver.12 (Genetyx) were used for aligning sequencing chromatographs to reference sequences.

Total cellular DNAs were prepared following Davis et al. (2002a (link)) or were obtained from other sources (see Acknowledgments). Voucher information is listed in Table S1.
Amplification and sequencing protocols for obtaining matK followed Cameron et al. (2001 (link)), using their primers 400F, trnK-2R, and 842F; ndhF followed Davis et al. (2001 (link)); rbcL followed Cameron et al. (2001 (link)); and PHYC followed Davis et al. (2002b (link)) with the addition of forward primer int-1F, which produced an ~800 base-pair (bp) amplicon when paired with reverse primer 623r/cdo (Davis and Anderson, 2010 (link)).
Double-stranded polymerase chain reaction (PCR) products were primarily gel extracted and purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, California, USA). PCR products were sequenced in both directions using dye terminators and sequencing protocols at the University of Michigan DNA facility (Ann Arbor, Michigan, USA), MWG Biotechnology (High Point, North Carolina, USA), and GENEWIZ, Inc. (Cambridge, Massachusetts, USA). Chromatograms were assembled into contiguous sequences and checked for accuracy using the software program Sequencher v4.7 (Gene Codes Corporation, Ann Arbor, Michigan, USA). All newly generated sequences were submitted to GenBank (see Table S1).

Phylogenetic inference was performed with each identified haplotypic group to examine the possibility of defining clades using the tree topology. Sequences of 482 bp segment of cyt b were aligned using Sequencher v4.7 (Gene Codes Corporation, Ann Arbor, MI, USA). The size of this DNA segment equivalent to 42.3% of the total gene is of common use as amplicon in studies of avian haemosporidia (Additional file 2). Eighteen very short centroid sequences were omitted from the alignment, because of the small size between 186 and 356 base pairs. To determine the phylogenetic relationships among the remaining haplotypes, 7 independent Bayesian analyses were performed with BEAST v1.8.3 [23 (link)] in the platform CIPRES Science Gateway V. 3. [24 ]. The Bayesian phylogeny was inferred from using a nucleotide substitution model GTR + G + I [25 ], a fixed substitution rate of 0.012 sequence divergence per a million years [26 (link)], and a posterior probability value of 0.95 as minimum clade support. The consensus tree was generated in PAUP4 [27 ] using a 25% burn-in; further, the consensus tree was visualized in the ITOL platform [28 (link)].

One isolate for each morphotype was used for genomic DNA extraction applying the standard cetyltrimethylammoniumbromide (CTAB)-based protocol [57 (link)]. According to the standard method for fungal identification, the internal transcribed spacer (ITS) regions of the ribosomal DNA were amplified and sequenced with primers ITS1/ITS4 [58 ]. PCR amplification and sequencing of amplicons was carried out as described by [59 (link)]. Sequences were edited with Sequencher v 4.7 (Gene Codes Corporation, Ann Arbor, MI, USA) and compared with sequences deposited in GenBank through BLASTn searches. New sequences were deposited in GenBank (Table 1). Sequences of Lulworthiales currently known from cultures were retrieved from GenBank (Table 2) and aligned with sequences of the isolates obtained in this study. Phylogenetic analyses were performed as described by [60 (link)]. Alignments were made using ClustalX v. 1.83 [61 (link)] and, if necessary, manually edited using MEGA6 [62 (link)]. Maximum likelihood (ML) analyses were performed on a Neighbour-Joining starting tree automatically generated by MEGA6. Nearest-Neighbour-Interchange (NNI) was used as the heuristic method for tree inference, and 1,000 bootstrap replicates were performed.

Population sequencing was undertaken using the Big Dye ready reaction terminator mix (V3) (Department of Zoology, University of Oxford) using gag-protease sequencing primers SQ2FC (CTT CAG ACA GGA ACA GAG GA), GF100-1817.18 (TAG AAG AAA TGA TGA CAG), gf2331 (GGA GCA GAT GAT ACA GTA TT), SQ16RC (CTT GTC TAG GGC TTC CTT GGT), GAS4R (GGT TCT CTC ATC TGG CCT GG), pan1dRx (CAA CAA GGT TTC TGT CAT CC), GR1981 (CCT TGC CAC AGT TGA AAC ATT T), and gr2536 (CAG CCA AGC TGA GTC AA). Sequence data were analysed using Sequencher v.4.8 (Gene Codes Corporation). Nucleotides for each gene were aligned manually in Se-Al v.2.0a11. Maximum-Likelihood phylogenetic trees were generated using PHYm131^{46 (link)} (http://www.hiv.lanl.gov) and visualised using Figtree^{47 (link)} v.1.2.2 (http://tree.bio.ed.ac.uk/software/figtee/).

For confirmation of the specificity of the amplification, all PCR products were cleaned up using Illustra ExoStar Kit (GE Healthcare, UK) and sequenced with both forward and reverse primers using the Big Dye Terminator Cycle Sequencing kit v3.1 (Life Technologies, USA), according to the supplier's protocol. Purification of the sequences from residual dye terminators was performed by Isopropyl alcohol 75%/Ethanol 70% precipitation. Capillary Electrophoresis of the sequences was carried out in an ABI3500 Genetic Analyzer (Life Technologies, USA). Electropherograms were visualized and edited with Sequencher v4.8 software (Gene Codes Corporation, USA). The obtained sequences were aligned with GeneBanks reference sequence.