Abi 3130xl

Bacterial community structures were monitored by amplifying the hypervariable regions V1–V3 of the 16S rRNA gene with the general bacterial primers fD1 and 5′FAM-labeled PRUN518r (Mikkonen et al. 2011 (link)). The volume of one PCR reaction was 50 μL, containing 2 μL undiluted DNA extract, 0.2 mM of each dNTP (Thermo Scientific), 0.3 μM of each primer (Oligomer), 0.7 mg mL⁻¹ BSA (Thermo Scientific), 1× PCR buffer (Biotools), and 1 U DNA polymerase (Biotools). The amplification was done in a DNA Engine DYAD^™ thermal cycler (MJ Research Inc., USA) with the following program: 5 min at 95 °C followed by 28 or 30 cycles of 45 s at 94 °C, 1 min at 55 °C, 1 min at 72 °C, and finally 5 min at 72 °C. The PCR products were checked by agarose gel electrophoresis (1.5 % agarose gel run at 100 V for 1 h). Fragment analysis by polyacrylamide capillary electrophoresis (ABI 3130 XL, Applied Biosystems) was outsourced to DNA Sequencing and Genomics Laboratory Core Facility, University of Helsinki, Finland, using GeneScan 600 LIZ (Applied Biosystems) as sizing standard.

The ex vivo retrotransposition assay was performed as follows. Approximately 3 × 10⁵ Sf9 cells in a 12-well plate were transfected with 800 ng of the target plasmid with the TransFast™ Transfection Reagent (Promega). Subsequently, these cells were infected with SART1Bm at MOI1. Plasmid DNA was extracted 72 h after infection. The PCR assay was conducted for SART1Bm with Ex-Taq (TaKaRa, Shiga, Japan). PCR was denatured at 94 °C for 1 min, followed by 35 cycles of 94 °C for 20 s, 60 °C for 30 s, and 72 °C for 50 s. The primers used for the nested PCR assay are shown in Supplementary Table 1. The PCR products were directly cloned into the pGEM-T Easy vector (Promega). The cloned products were sequenced with a BigDye Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) on ABI 3130xl and 3500/3500xl Genetic Analyzers (Applied Biosystems). Sequence analysis was performed using the Vector NTI Advance 10 system (Invitrogen).

Two Brazilian field isolates BI/BR/Embrapa/331/2000 (accession number: KU727196, identified as IBV A isolate) and BI/BR/Embrapa/127/2006 (accession number: KU727200, identified as IBV B isolate), were isolated from clinical cases of avian infectious bronchitis in poultry flocks located in southern Brazil. Virus isolation and titration were carried out by inoculation of five 10-day-old specific pathogen-free (SPF) embryonated chicken eggs/sample via the allantoic sac route [24 (link)] and virus titers were expressed as 50% embryo infectious doses (EID₅₀) according to the Reed & Muench method [25 ]. IBV field strains were genotyped by automated DNA sequencing (ABI3130XL, Applied Biosystems) of a 1059 bp portion of the S1 gene amplified as described [26 (link)]. The deduced amino acid sequences of S1 protein of IBV A and B isolates have 93.16% of identity, and with regard H120 strain (Massachusetts serotype) of IBV they have 69.4% and 70.0% identity, respectively.

The coding exons of WFS1 (NM_006005.3) and CISD2 (NM_001008388.4) were amplified with intronic primers by using standard conditions. Primers and PCR conditions are available on request. PCR products were purified with an Illustra ExoProStar enzyme (GE Healthcare, England), processed with a BidDye® Terminator Cycle Sequencing kit (Thermo Fischer, Foster City, CA, USA) and analyzed on an ABI 3130 XL automated sequencer (Applied Biosystems).

Prioritized variants were independently validated by Sanger sequencing. PCR products were either directly sequenced using GENEWIZ, ABI 3130XL, and BigDye v3.1 Terminators (Applied Biosystems) per the manufacturer’s protocols or sequenced after gel purification using the MinElute Gel Extraction Kit (QIAGEN). Sequencing primers are listed in S7 Table. All compound heterozygous variants described in the main text were confirmed on different alleles (phased) using sequenced, cloned gDNA or cDNA derived from the patients’ fibroblasts. Patients’ familial DNA was also sequenced for haplotype phasing when available. All information about the experimentally confirmed localization of compound variants within a separate allele is presented in S3 Table.

The 16 microsatellite markers described in Puppo et al. [31 (link)] were amplified using the same multiplex primer combinations, fluorescent dyes, and PCR conditions from Puppo et al. [23 (link), 31 (link)]. Genotyping was done in an ABI3130xl automatic sequencer (Applied Biosystems, Inc., Foster City, CA; USA) using an internal size standard (Genescan-500 LIZ; Applied Biosystems, Inc.). GeneMapper ver. 4.0 was used for allele scoring (Applied Biosystems, Inc.). A total of 96 individuals were genotyped two times to evaluate scoring consistency. After scoring, only markers showing data for a majority of the samples were used for further analysis. This led to the exclusion of one marker (6493). Puppo et al. [23 (link), 31 (link)] did not find any significant deviation from the Hardy-Weinberg equilibrium, genotyping errors or a high amount of null alleles for any of the markers, so no further exclusions were necessary.