As a soil sample can contain cysts from different Heterodera species, we used restriction profiles of the ITS sequence for species identification. DNA extraction, PCR amplification of ITS sequence and digestion of PCR products were performed as described in Amiri, Subbotin, and Moens (2002 ). Polymorphic mitochondrial markers are not yet described in H. schachtii, but we used nuclear microsatellite loci that are markers of choice for studying neutral genetic structure. In all, 1,754 H. schachtii individuals were identified and successfully genotyped at eight microsatellite loci, named Hs33, Hs36, Hs55, Hs56, Hs68, Hs84, Hs111, and Hs114 and described in Montarry et al. (2015 ). Microsatellites PCR products were analyzed on an ABI Prism® 3130xl sequencer (Applied Biosystems, Foster City, CA, USA). Allele sizes were identified using the automatic calling and binning procedure of GeneMapper v4.1 (Applied Biosystems) and completed by a manual examination of irregular results. Samples with dubious genotypes were reamplified.
Abi prism 3130xl sequencer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI PRISM 3130xl sequencer is a capillary electrophoresis-based genetic analyzer designed for DNA sequencing and fragment analysis applications. It features 16 capillaries and is capable of generating high-quality sequencing data.
Automatically generated - may contain errors
Lab products found in correlation
Genemapper v4, by Thermo Fisher Scientific (2 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (2 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (2 mentions)
Abi prism 3.1 bigdye terminator kit, by Thermo Fisher Scientific (2 mentions)
Taqman pre designed snp genotyping assay, by Thermo Fisher Scientific (1 mentions)
Taqman snp genotyping, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator cycle sequencing kit version 1, by Thermo Fisher Scientific (1 mentions)
Maldi tof, by Labcorp (1 mentions)
Pcr purification kit, by Roche (1 mentions)
Bigdye terminator ver 3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Abi bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Zero blunt topo pcr cloning kit, by Thermo Fisher Scientific (1 mentions)
Exosap it, by Thermo Fisher Scientific (1 mentions)
Genemarker, by SoftGenetics (1 mentions)
Bigdye terminator cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Abi prism bigdye terminator v3.1 kit, by Thermo Fisher Scientific (1 mentions)
Kod plus dna polymerase, by Toyobo (1 mentions)
Abi prism 3.1 big dye terminator chemistry, by Thermo Fisher Scientific (1 mentions)
Sequencher 4, by Gene Codes (1 mentions)
Sequencer 4, by Gene Codes (1 mentions)
18 protocols using abi prism 3130xl sequencer
1
Genetic Identification of Cyst Nematode Species
2
Genotyping Cyst Nematode Populations
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For each sampled nematode population, 40 second‐stage juvenile larvae were used to perform DNA extraction and each larva was extracted from a different and randomly chosen cyst, to avoid family structure biases caused by sibling relationships. A soil sample can contain cysts from different Heterodera species; therefore, molecular characterization based on the restriction profiles of ITS sequence was used for species identification. By multiplying the ratio of H. schachtii among the 40 larvae with the number of cysts we sampled, we estimated the number of H. schachtii cysts contained in our samples. DNA extraction, PCR amplification of ITS sequence, and digestion of PCR products were performed as described in Gracianne et al. (2014 ).
We ultimately genotyped 761 and 854 H. schachtii individuals in 2012 and 2013, respectively, using eight microsatellite loci, named Hs33, Hs36, Hs55, Hs56, Hs68, Hs84, Hs111, and Hs114 and described in Montarry et al. (2015 ). Microsatellites PCR products were analyzed on an ABI Prism® 3130xl sequencer (Applied Biosystems™, ThermoFisher Scientific, Waltham, MA, USA), and allele sizes were identified using the automatic calling and binning procedure of GeneMapper v4.1 (Applied Biosystems™), with any irregular results evaluated manually. Samples with dubious genotypes were reamplified.
We ultimately genotyped 761 and 854 H. schachtii individuals in 2012 and 2013, respectively, using eight microsatellite loci, named Hs33, Hs36, Hs55, Hs56, Hs68, Hs84, Hs111, and Hs114 and described in Montarry et al. (
3
SNP Genotyping from Peripheral Blood
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Genomic DNA was extracted from peripheral blood leukocytes according to established protocols. Genotyping of SNPs was carried out by direct sequencing, TaqMan SNP genotyping (Applied Biosystems, Foster City, USA) or by primer extension of multiplex PCR products and subsequent allele detection by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF; Sequenom, San Diego, USA). Direct sequencing was performed with the Big Dye Terminator Cycle sequencing kit version 1.1 (Applied Biosystems, Foster City, U.S.A.) according to the manufacturer’s instructions. Reactions were analyzed with an ABI Prism 3130xl sequencer (Applied Biosystems). TaqMan pre-designed SNP genotyping assays (Applied Biosystems) were used according to the manufacturer’s instructions. The rs144087548 variant was genotyped by polymerase chain reaction (forward primer: 5′-CGC AGA CAT GAT GCT GGG GGT-3′; reverse primer: 5′-ACA TGC AAG ACG GGG AAT TGA-3′) followed by HpyCH4III digestion (New England Biolabs, Ipswich, USA) and restriction fragment length analysis. All SNPs showed high genotyping quality with an average call rate >98 % in each of the five case–control samples.
4
Mitochondrial COI Sequencing of Pigeons
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Mitochondrial COI sequence was analyzed for one sample of the 10 Egyptian breeds and of Japanese feral pigeons. Moreover, one sample was sequenced from the four wild pigeon species: Emerald dove (Chalcophaps indica), Oriental turtle dove (Streptopelia orientalis), Whistling green pigeon (Treron formosae), and white-bellied green pigeon (Treron sieboldii) for comparison. The primers and polymerase chain reaction (PCR) condition are the same as described by Ramadan et al. [10 ].
The amplified products were purified using PCR Purification Kit (Roche, Mannheim, Germany), and the resultant products were sequenced using the same primers and the Big Dye Terminator ver. 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) according to the standard protocol and electrophoresed on an ABI PRISM 3130xl sequencer (Applied Biosystems). The MEGA 6 Software (https://www.megasoftware.net ) [16 (link)] was used for sequences alignment and to infer the phylogenetic relationships based on neighbor-joining [17 (link)] methods [18 (link)].
The amplified products were purified using PCR Purification Kit (Roche, Mannheim, Germany), and the resultant products were sequenced using the same primers and the Big Dye Terminator ver. 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) according to the standard protocol and electrophoresed on an ABI PRISM 3130xl sequencer (Applied Biosystems). The MEGA 6 Software (
5
NLRP5 Sanger Sequencing for Variant Confirmation
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Sanger sequencing of NLRP5 was used to confirm exome variants, establish their inheritance, fill gaps in exome coverage and screen a further 14 patients (four BWS–MLID, five SRS–MLID, three transient neonatal diabetes mellitus–MLID and two idiopathic-MLID) and 19 mothers of individuals with MLID.
M13 universal tagged primers were designed to 14 of the 15 exons of NLRP5 (seeSupplementary Table 4 ). Exons were amplified using Q5 High-Fidelity DNA Polymerase (New England BioLabs). Amplicons were then treated with ExoSAP to degrade any remaining primers, before sequencing with M13 forward and reverse primers using BigDye 1.1chemistry (Applied Biosytems). Sequencing reactions were analysed on an ABI Prism 3130XL sequencer (Applied Biosystems).
Co-amplification of exons 5 and 6 was observed with both primer sets due to their highly similar sequence and required the use of internal sequencing primers with exon-specific terminal-3′ bases (seeSupplementary Table 4 ) to generate exon-specific sequencing. Repetitive sequence at Exon 4 required the use of primers (see Supplementary Table 4 ) without M13 universal tags and Phusion High-Fidelity DNA Polymerase (New England BioLabs) to generate the sequencing template. Exon 4 amplicons were sequenced as previously described using the amplification primers.
M13 universal tagged primers were designed to 14 of the 15 exons of NLRP5 (see
Co-amplification of exons 5 and 6 was observed with both primer sets due to their highly similar sequence and required the use of internal sequencing primers with exon-specific terminal-3′ bases (see
6
Genetic Analysis of Hearing Loss Genes
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Informed consent was obtained from patients and parents according to current national rules and laws. Molecular genetic studies of the GJB2, GJB6, and SLC26A4 genes and mitochondrial DNA (mit-DNA) were performed in 77 patients. Genomic DNA was extracted by standard protocols from peripheral blood leukocytes of patients. Direct DNA sequencing of the GJB2 gene (including analysis of the entire coding region) was performed. PCR amplification of the coding 21 exons, the flanking, and promoter regions of the SLC26A4 gene was performed using specific primers. Amplification reactions were performed in a final volume of 25 mL containing 100 ng of genomic DNA, 200 mmol/L dNTPs, 10 mmol/L each primer 1.5 mmol/L MgCl2, and 1 U of Taq polymerase. After 5 min of denaturation at 94°C, 35 PCR cycles were carried out, each cycle comprising 45 s of denaturation at 94°C, 45 s of annealing at 60°C, and 80 s of extension at 72°C. Direct sequencing of the PCR products on both strands was performed on an ABI PRISM 3130xl sequencer, using the ABI BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems by Life Technologies).
7
16S rRNA Gene Sequencing from Enrichment Cultures
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PCR reactions from enrichment cultures were performed with primers 27f and rd1 (5′-AAGCTTAAGGAGGTGATCCAG-3′), adapted from [31 (link)]. Zero Blunt TOPO PCR cloning kit (Invitrogen) was used for cloning obtained 16S rRNA gene PCR amplicons. Selection of individual clones for sequencing was based on AluI or HhaI restriction profile polymorphism of PCR amplicons obtained from cloned fragments with T3 and T7 universal primers, and visualized on NuSieve 3:1 Agarose gels (2% equivalent agarose; Tebu-bio, Le Perray-en-Yvelines, France). Sequences of PCR products, purified with ExoSAP-IT (USB Corporation, USA) following the manufacturer’s protocol, were obtained with primers 27f, 534r or rd1 on an ABI Prism 3130 XL sequencer (Applied Biosystems, Waltham, MA, USA). Similarity searches of the 16S rRNA gene sequences were performed using Blast and the Ribosomal Database Project [32 (link)].
8
Microsatellite Profiling of Phytophthora ramorum
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The population structure of P. ramorum NA1 was first reconstructed using microsatellite markers [34 (link),35 (link),36 (link)] and were compared to results from the SNP phylogeny. The 42 isolates were genotyped using six microsatellite loci (Table S2 ), PrMS39a, PrMS39b, PrMS45, PrM43a and PrMS43b [40 (link)] and locus 64 [41 (link)] using primers and thermal cycling conditions previously described [36 (link),40 (link),41 (link)]. Successful PCR amplifications were verified on 1.5% agarose gels and PCR products were subsequently sized on an ABI PRISM 3130xl sequencer (Applied Biosystems, Foster City, United States) using Rox 500 as size standard. Allele size assignments were performed using Genemarker (SoftGenetics LLC, State College, United States). Allelic data were formatted for the program GenAlex6 [42 (link)] which was used to identify identical MLGs among the 42 isolates. To illustrate the genetic variation among unique MLGs, a neighbor-joining tree was constructed based on Nei’s genetic distance, Da [43 (link)] using the program POPULATIONS 1.2.30 [44 ]. The tree was visualized using Nei’s genetic distance to identify points of reticulation. The microsatellite network was visualized on the program SplitsTree4 [45 (link)].
9
Targeted Sanger Sequencing for Methylation-Dependent SNPs
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The presence and correct position of methylation-dependent single-nucleotide variant C/T SNPs in the synthetic DNA sequences was verified by targeted Sanger sequencing. Briefly, sequencing analyses (DNA and bisulfite sanger sequencing) were performed using BigDye® Terminator Cycle Sequencing kit (Applied Biosystems) on an ABI Prism 3130XL sequencer (Applied Biosystems), following standard procedures.23 (link)
10
Genetic Engineering Techniques in Bacterial Systems
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Established methods were employed for the preparation of plasmids and genomic DNAs, their digestion with restriction endonucleases, ligation, and agarose gel electrophoresis, and the transformation of E. coli cells.28 ,33 Electroporation of sphingomonad strains was performed as described previously.34 (link) PCR for cloning was performed with KOD-Plus DNA polymerase (TOYOBO, Osaka, Japan). The primers used are listed in Supplementary Table S2 . The Sanger sequencing was performed using an ABI PRISM 3130xl sequencer and ABI Prism Big Dye Terminator v3.1 Kit (Applied Biosystems).
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