Hiscan platform

Genomic DNA was extracted from blood samples using standard phenol-chloroform procedures and was initially quantified using spectrophotometry. Prior to hybridization to the chip, all samples were re-quantified using pico-green fluorimetry and DNA quality was assessed using standard gel electrophoresis techniques; this ensured that only samples of high quality DNA were used. Automated procedures were used to hybridize the cases and controls to the HumanCoreExome-12v1_A Beadchip (Illumina Inc., San Diego CA, USA) and scanned on the Illumina HiScan platform, using standard protocols. This particular chip comprises of 547644 markers, including all the tag markers (264,909 markers) on the HumanCore beadchip, over 240,000 markers of the HumanExome chip as well as several rare variants.

DNAm analysis was performed at the Genetics Core of the Edinburgh Clinical Research Facility (Edinburgh, UK). Bisulphite conversion of 500 ng input DNA was carried out using the EZ DNAm Kit (Zymo Research, Freiburg, Germany). Four microlitres of bisulphite-converted DNA was processed using the Infinium HD Assay for Methylation (Illumina Methylation 450k beadchip and Infinium chemistry (Illumina, San Diego, CA, USA)). Each sample was interrogated on the arrays against 485 000 methylation sites. The arrays were imaged on the Illumina HiScan platform and genotypes were called automatically using GenomeStudio Analysis software version 2011.1 (Illumina). The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE72120 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE72120).

We used a poly A selection protocol coupled with the Illumina TruSeq RNA-Seq library protocol to construct the mouse Bodymap RNA-Seq libraries. To monitor the quality of the transcriptomic profile data in the RNA-seq experiments, external RNA Control Consortium (ERCC) spike-in controls^{45 (link)} were added in each sample in an amount equivalent to about 1% of the mRNA content in the total RNA sample being sequenced before library construction. One single RNA-seq library was constructed for each RNA sample. Each library was sequenced using an Illumina HiScan platform (101 bp pair-end read). Eighteen different libraries (biological samples, randomized) were pooled together in equal amount and loaded onto two lanes of a flow cell (Supplementary Table S1), which would make two technical replicates from each biological sample. Reads from the two technical replicates of the same sample were combined together before we conducted the analyses.

Tissue biopsies were obtained from two male bowhead whales harvested by Inupiat hunters at Barrow, Alaska during the Fall hunt of 2010; heart, cerebellum, liver, and testes were biopsied from male bowhead number 10B16, and retina from male bowhead 10B20. Samples were immediately placed in liquid nitrogen and transported in a dry shipper to Purdue University. RNA was extracted using TRIZOL reagent (Invitrogen) following the manufacturer’s protocol. RNA was purified using an Invitrogen PureLink Micro-to-Midi columns from the Total RNA Purification System using the standard protocol. RNA quantity and quality was estimated with a spectrophotometer (Nanodrop) and by gel electrophoresis using an Agilent model 2100 Bioanalyzer. cDNA libraries were constructed by random priming of chemically sheared poly A captured RNA. Randomly primed DNA products were blunt ended. Products from 450–650 bp were then isolated using a PippenPrep. After the addition of an adenine to the fragments, a Y primer amplification was used to produce properly tailed products. Paired-end sequences of 100 bp per end were generated using the Illumina HiScan platform. Sequences with primer concatamers, weak signal, and/or poly A/T tails were culled. The Trinity software package for de novo assembly (Grabherr et al., 2011 (link)) was used for transcript reconstruction (Table S1).

Genomic DNA was enriched for selected RNA-binding proteins and known ALS genes using a custom designed Agilent SureSelect in solution kit. Sequencing was performed using an Illumina HiScan platform according to manufacturers instructions.
Rare deleterious mutations were defined by frequency within the Exome Aggregation Consortium data set of <1/10,000 control alleles (Lek et al., 2016 (link)), and a Phred-scaled Combined Annotation Dependent Depletion (CADD) score >10 (Kircher et al., 2014 (link)). Comparison of various pathogenicity prediction tools recently supported the sensitivity and specificity of CADD (Salgado et al., 2016 (link)). Given that we were focused on exonic changes with an effect on protein function, synonymous changes were excluded. We excluded any changes with a read depth <10 and validated by Sanger sequencing any changes with read depth 10–15 or a novel allele frequency less than one third the reference allele frequency (Supplementary Figure 1).
ExAC defines constrained genes based on an observed frequency of loss of function mutations which is much less than predicted by sequence specific mutation probabilities (Lek et al., 2016 (link)). A threshold for “constrained” is set as probability of a gene being loss of function intolerant (PLi) > 0.95.