Kingfisher duo prime

The poliovirus detection assay was developed specifically for this study and was a multiplex 2-step reverse transcription, quantitative polymerase chain reaction assay (RT-qPCR). Ribonucleic acid was extracted from frozen stool samples using the MagMAX Viral RNA Isolation Kit on the Kingfisher Duo Prime instrument (both from Thermo Scientific Inc.), and reverse transcription utilized the SuperScript III Reverse Transcriptase (Thermo Fisher Scientific Inc.). Real-time polymerase chain reaction was performed in a total volume of 20 microliters, consisting of 5 microliters of complementary deoxyribonucleic acid (cDNA) and 15uL of iQ Multiplex Powermix with Sabin 1, 2, and 3 specific primers and probes.
Amplification and detection was performed on the CFX384 Real-Time System (BioRad). Samples were run in triplicate, and a sample was considered positive if 2 out of 3 reactions crossed the cycle threshold (C_T) in less than 37 cycles. To account from sample handling variability during real-time polymerase chain reaction testing, Sabin 1, 2 and 3 positive controls, a negative control, and No Template Control (NTC) were then run together on 384 well plates with the unknown samples above.
Finally, genetic sequencing using the Sanger technique was performed on these OPV-positive samples to rule out the possibility of false positives due to other enteroviruses [4 (link)].

Processed samples were initially tested for the presence of coronavirus using a pancoronavirus nested PCR targeting the RdRp gene as previously published (Table S2) (16 (link)). Briefly, 100 μL of each sample was used to extract RNA using the MagMAX core nucleic acid purification kit (Applied Biosystems, Carlsbad, CA, USA) and KingFisher duo prime (Thermo Scientific, Carlsbad, CA, USA), according to the manufacturers’ instructions. Purified RNA was used to perform the first round of RT-PCR using the Quanta qScript XLT one-step RT-PCR kit (Quantabio, Beverly, MA, USA). The first-round product was used for a second PCR using Dream Taq green master mix (Thermo Scientific, Carlsbad, CA, USA). The final product of 440 bp was gel purified using a MEGAquick-spin plus kit (iNtRON, Seoul, South Korea) and analyzed by Sanger sequencing. In addition, purified RNA was tested for the presence of BCoV RNA by targeting the BCoV nucleocapsid gene as previously described (Table S2) (15 (link), 40 (link)). Briefly, purified RNA was used to perform RT-qPCR using the Quanta qScript XLT one-step RT-qPCR ToughMix kit (Quantabio, Beverly, MA, USA) and analyzed using the Bio-Rad CFX 96 real-time detection system (Bio-Rad, Hercules, CA, USA).

A peripheral blood sample was obtained from the patient and her father, and placed in EDTA tubes. Automated extraction of genomic DNA was performed using the KingFisher Duo Prime purification system (Thermo Fisher Scientific, Waltham, MA, USA). The patient’s DNA was analyzed by whole exome sequencing (WES), focusing on a panel of 30 genes related to LCA (Table S1), and the results were filtered according to deleterious potential and minor allele frequency, as previously reported [23 (link)]. Putative pathogenic variants were confirmed by Sanger sequencing. Moreover, whole genome sequencing (WGS) of the patient’s DNA was also performed using the Illumina platform and the TruSeq DNA PCR-Free library kit (Illumina, San Diego, CA, USA), obtaining an average depth of 39× and a fragment length median of 390 bp. The study of gross genomic rearrangements within the LCA genes was carried out by analyzing sequencing reads in BAM files from WES and WGS with the Alamut Visual software v2.11 (SOPHiAGENETICS, Boston, MA, USA). Moreover, PCR confirmation of the NMNAT1 exon 4 and 5 duplication was performed using a forward primer located in the 3′UTR and a reverse primer matching the intron 3 (Table S3). Finally, the familial origin of the detected pathogenic variants was established based on segregation analyses using the DNA sample from the father.

Intracranial GCT tissues were obtained from surgical resection and immediately fixed with formalin. The tissues were then embedded with paraffin and stored. Prior to DNA extraction, the FFPE sample was dewaxed, digested with protease K, and then vortexed to decrosslinking.
Bone marrow samples were collected from the AML-M7 patient and DNA was isolated using the MagMAX™ DNA extraction kit (A36570, Thermo Fisher Scientific) using Nucleic acid extractor KingFisher Duo Prime (Thermo Fisher Scientific). The DNA was then assessed for quality using a Nanodrop 2000.

We obtained 354 tissue samples collected by the California Department of Fish and Wildlife between 2011–2017 from pumas either hit by car (~6%), found dead (~2%), poached (<1%), or through depredation permits (>90%), which had never been used in any previous genetic survey. Samples were well‐distributed throughout the state, except for smaller populations in smaller mountain ranges. To bolster our sample size in the Los Angeles region of southern California, we added the only remaining DNA extracts (N = 144) from pumas collected between 2002–2015 (Riley et al., 2014 (link); Vickers et al., 2015 (link)). After genomic and bioinformatic filtering (described below), we retained 401 out of 498 samples in the final dataset, which spanned the majority of puma habitat in California, excluding desert regions (Figure 1). For samples that lacked a precise GPS location, we used the nearest address or town where they were collected as their GPS point. Samples were stored at −80°C until DNA was extracted using Omega Bio‐tek Mag‐Bind Blood & Tissue DNA HDQ Kits (Omega Bio‐tek, #M6399‐01), with a manufacturer‐designed protocol for the Kingfisher Duo Prime (ThermoFisher Scientific, #5400110) automated DNA purification system. We measured the concentration of DNA from each sample using a Qubit 3.0 fluorometer (Invitrogen, #Q33216) with Qubit dsDNA high‐sensitivity kits (Invitrogen, #Q32854).