Sequencher software

Phylogenetic trees were generated based on the hemagglutinin (HA) gene sequence. For direct sequencing of viral nucleic acids from clinical specimens, gene fragments were amplified and sequenced with the use of Big Dye terminator cycle sequencing kit (version 3.1, Applied Biosystems, Foster City, CA, USA) on an ABI 3130XL DNA sequencer (Applied Biosystems, Foster City, CA). Nucleotide sequences of PCR products were analyzed using Sequencher software (version 4.8, Gene Codes Corporation) and then aligned with the CLUSTAL X version 2.1 software and BioEdit (version 7.2.3 -Isis Pharmaceuticals, Inc.) software to compare with influenza sequences from the GenBank database. Phylogenetic trees were developed using MEGA (version 5.2.2), the analysis was inferred using the neighbor-joining method, and the evolutionary distances were computed using the Kimura 2-parameter method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches.

The entire coding region of CD59 gene (transcript variant CD59-211 ENST00000642928.1) was amplified by polymerase chain reaction (PCR) and Sanger sequenced on an ABI 3100 automatic sequencer (Applied Biosystems, Foster City, CA, USA).
Resulting electropherograms were visually analyzed using Sequencher software (Gene Codes Corp. Ann Arbor, MI, USA). Primer pair sequences and PCR conditions are available under request. In silico evaluation of potential deleterious effects of CD59 genetic variants was performed with the CADD-score (http://cadd.gs.washington.edu/), Mutation Taster (http://www.mutationtaster.org/), SIFT (http://sift.jcvi.org/), and Human Splicing Finder tool (http://www.umd.be/HSF3/index.html).

Dried blood spots were collected during the phase II FMP2.1/AS02_A vaccine trial from both routine monthly clinic visits, and unscheduled sick visits. Samples from microscopically-detectable P. falciparum infections underwent DNA extraction and PCR amplification of ama1^{21 (link)}. Following capillary sequencing, Sequencher software (Genecodes) was used to identify single and predominant clone infections, and unique ama1 haplotypes were then amplified using a specific primer-linker sequence that facilitated cloning into a pXI plasmid vector. Cloning, transformation, and amplification protocols have been described^{46 (link)}. Single-clone ama1 haplotype translation was ensured by use of a single colony amplification protocol^{25 (link)}. Two hundred and sixty-three unique ama1 whole-protein sequences were observed among 412 sequences analyzed. Protein microarray printing, probing, data generation, and quality control protocols have been described^{46 (link)}.

Following NGS sequencing, EGFR and KRAS (including codons 12, 13) mutations in samples from the RCRC set as well as KRAS mutations from RSCRR set were validated either by Sanger sequencing or Real-Time PCR. Samples from RSCRR set were pre-screened for EGFR mutations and, thus, not required further validation with orthogonal methods. EGFR mutations (including exons 18–21) were examined by Sanger Sequencing. Primer pairs were 5′-CTGAGGTGACCCTTGTCTCTG-3′ and 5′-CCAAACACTCAGTGAAAC-3′, 5′-TGCCAGTTAACGTCTTCCTT-3′ and 5′-CAGGGTCTAGAGCAGAGCAG-3′, 5′-CATTCATGCGTCTTCACCTG-3′ and 5′-TTATCTCCCCTCCCCGTATC-3′, and 5′-TGATCTGTCCCTCACAGCAG-3′ and 5′-GGCTGACCTAAAGCCACCTC-3′ for exons 18, 19, 20 and 21 respectively. Thermal cycling conditions included 5 min at 95 °C followed by 35 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min and one cycle of 72 °C for 7 min. The PCR products were further purified with USB ExoSapit (GE Healthcare, Uppsala, Sweden) followed by cycle sequencing with the BigDye Terminator version 3.1 cycle sequencing kit (Applied Biosystems) according to the manufacturer’s protocol and resolved on an ABI 3500xL sequencer (Applied Biosystems). Sequence chromatograms were analyzed by Sequencher software (Gene Codes Corp, Ann Arbor, MI), followed by manual review. KRAS codon 12, 13 mutations were examined by pyrosequencing as described previously [33 (link)].

DNA was extracted from whole blood using a standard salting-out method. SNX10 gene amplification and bidirectional sequencing of exons and intron-exon boundaries were performed in samples from all patients and their parents. RNA was extracted from cultured patient peripheral blood monocytes and converted to cDNA as described below for gene expression analysis. Bidirectional Sanger sequencing (Applied Biosystems BigDye Terminator v3.1 Cycle Sequencing Kit, Applied Biosystems) on a 3730xl DNA Analyzer (Applied Biosystems) was used for analysis of SNX10 cDNA (NM_001199837.1) using primer in exon 3 to 5 to confirm the mutation and to identify the effect of the splicing. Sequence analysis was performed using the Sequencher software (Gene Codes Corporation, Ann Arbor, MI, USA). Primers sequences where designed using Primer 3 Plus software (primer3plus.com/) and are available upon request. Five patients (Pt1–5) were analyzed using the GeneChip Mapping 250 K array (Affymetrix, Santa Clara, CA), and one patient (Pt9) was analyzed using the HumanOmniExpressExome-8v1array (Illumina), for haplotype analysis, and estimation of the age of the sequence variant. To calculate the carrier frequency, DNA from 1000 randomly chosen individuals from the county of Västerbotten was used (The medical bio-bank, Umeå university hospital, Umeå, Sweden)^{50 (link)}.