Proton p1 chip

Mice were sacrificed 3 days post-infection (dpi) and both lungs were extracted and transferred immediately to RNAlater (Qiagen), stored at 4°C for one day, and then stored at −20°C. RNA was isolated using Qiagen Midi kit (30 (link)). RNA quality was evaluated on a 2100 Bioanalyzer (Agilent). Five-hundred nanograms of total RNA was used to prepare libraries for sequencing using the Lexogen SENSE RNA-seq library kit for Ion Torrent. Libraries were amplified for 11 cycles as the final step of library preparation. Before sequencing, 1-µl aliquots were pooled and sequenced on an Ion Torrent PGM 314 chip. Barcoded data from the PGM was used to balance the final pool before sequencing. Library pools were sized to ~260 bp on a Pippin Prep instrument using 2% Pippin agarose gel. The sized libraries were evaluated on an Agilent High Sensitivity chip, quantified using real-time PCR, and used to prepare beads using a One-Touch 2 device. Beads were sequenced on an Ion Torrent Proton P1 chip. On average, 67 million reads were obtained per strain.

Whole exome sequencing of the Australian AML cohort has been previously reported^{22 (link)}. Rare complex I variants were identified as those with a minor allele frequency < 0.005 as reported in dbSNP137, 1000 Genomes (April 2012, any ethnicity) or the National Heart, Lung, and Blood Institute Exome Sequencing Project (evs-6500, any ethnicity). For Ion-Torrent sequencing, primers were designed for a custom amplicon panel using Ion Torrent’s AmpliSeq Designer (version 4.24) targeting the nuclear-encoded complex I genes. All primer sequences are listed in Supplementary Data 6. Libraries were sequenced on the Ion-Torrent Proton P1 chip aiming for >1000 × mean coverage. Sequences were aligned to the hg19 reference genome using Torrent Mapping Alignment Programme and variants were called as part of Torrent Suite v4.0 using stringent settings. Variants were filtered based on design content, read quality and strand bias. Filtered variants were annotated using an in-house pipeline. Variants were confirmed by Sanger sequencing. For all variants identified in the Australian cohort, population variant frequencies from the latest version of gnomAD are provided in Supplementary Data 1 and 3. For Stanford Hospital samples, exome sequencing has been previously reported^{28 (link)–31 (link)}.

Whole blood for circulating tumor DNA (ctDNA) analysis was collected at baseline and on treatment ± 5 days of cycle 3 day 1 from consenting patients to test the correlation of ctDNA response to clinical response. Whole blood draws (∼8 mL) were collected and processed to plasma by centrifugation at 1,600 × g for 10 minutes followed by a second spin at 16,000 × g for 10 minutes. Plasma was collected and frozen at −80°C. Two milliliters of plasma was used to isolate cell-free DNA (cfDNA) with the Qiagen circulating nucleic acid isolation kit (#55114) according to manufacturer's protocols. Following quantitation of the cfDNA using a Qubit fluorimeter, 10 ng of cfDNA was used to generate sequencing libraries with the Ion Torrent, Ion AmpliSeq Library kit and Ion AmpliSeq Cancer Hotspot V2 primer panel according to the manufacturer's protocols. Libraries were sequenced on either the Ion Torrent Proton P1 chip or the Ion Torrent Personal Genome machine 318 chip with a minimum average coverage of >1,200×. Variants were called using Ion Reporter software (v5.12) and filtered for known pathogenic and/or likely pathogenic mutations.