Abi 3130xl capillary sequencer

Oligo primers were designed for amplifying a target lesion of PIK3R1 (forward; 5′-ATTGCATGGAATTGTGAACTAATGC-3′, reverse; 5′-TGTTCTTAGGCAGTGCCACTTCA-3′). The Pfu DNA polymerase (Agilent technologies) and optimized thermal conditions were used for polymerase chain reaction (PCR). The PCR products were analyzed using the Big Dye Terminator kit (Applied Biosystems, CA, USA) and an ABI 3130xl capillary sequencer (Applied Biosystems).

To verify the genotype of rs671, Sanger sequencing was also carried out as our reference method using ABI 3130xl capillary sequencer (Applied Biosystems, Foster City, CA). The sequences of primers are shown in Table 1. The sequence was detected by Chromas program (Technelysium Pty. Ltd, Helensvale, Queensland, Australia).

Morphological observations and nutrients utilization tests of the two soil microbes, S223 and S224 were performed according to the procedures described in the International Streptomyces Project (Shirling and Gottlieb, 1966 (link)). Genomic DNA of the actinomycetes were extracted following standard DNA isolation protocol (Sambrook et al., 2012 ). The universal eubacterial primers: 8F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were used for the amplification of 16S ribosomal DNA gene (Salaun et al., 2010 (link)). Purified DNA preparations were run in ABI 3130xl capillary sequencer (Applied Biosystems) at the Station Biologique de Roscoff, Genopole Ouest, Roscoff, France. The 16S rDNA sequences were edited using DNAMAN v. 4.15 software, and compared with sequences deposited in public databases using BLAST (Basic Local Alignment Search Tool).

Eighty‐four individuals of T. delaisi were caught with hand nets at 10 localities in Croatia between 2006 and 2012 (Fig. 1, Appendix). Fin clips (small parts of the caudal fin) were taken and preserved in 96% ethanol, and fish were immediately released. Whole genomic DNA was extracted following a rapid Chelex protocol (Richlen and Barber 2005). The most variable part of the mitochondrial control region was amplified and sequenced according to the protocols described in Koblmüller et al. (2011) and Duftner et al. (2005), respectively. The primers used for PCR and chain termination sequencing were L‐Pro‐F_Tropheus (Koblmüller et al. 2011) and TDK‐D (Lee et al. 1995). DNA fragments were purified with Sephadex^™ G‐50 (GE Healthcare, Vienna, Austria) and visualized on an ABI 3130xl capillary sequencer (Applied Biosystems, Vienna, Austria). Additional sequences from Portugal, Italy, Cyprus, Croatia and the Azores and Canary islands (Domingues et al. 2007) were obtained from GenBank (Fig. 1, Appendix). Sequences were aligned by eye (no indels were present in the alignment) in mega 6.06 (Tamura et al. 2013). The length of the final alignment was 352 bp. Sequences are deposited in GenBank under the accession numbers KT267998–KT268081.

Blood samples from the individuals affected were obtained for genetic examination. Genomic DNA was extracted from peripheral blood using the conventional salting-out procedure. The entire coding region encompassing 15 exons and flanking intronic sequences of the CHM gene was amplified with PCR and directly sequenced in all the patients. The primers used for amplification and sequencing, as well as the PCR conditions are available upon request. The polymerase chain reaction (PCR) products were purified with the use of ExoSAP-IT (Exonuclease I and Shrimp Alkaline Phosphatase Cleanup for PCR products, Affymetrix) and directly sequenced using Dye Terminator chemistry (v3.1BigDye® Terminator, Life Technologies). The sequencing products were separated on an ABI 3130xl capillary sequencer (Applied Biosystems). The sequences obtained were verified by comparing them to the reference sequence of the CHM gene (GenBank NM_000390.2) and screened for mutations. Any variations identified were referred to the Human Gene Mutation Database (HGMD), Exome Variant Server (EVS), ExAC Browser Beta (Exome Aggregation Consortium 2015) and gnomAD browser beta (genome Aggregation database) for the CHM gene. Novel variants identified in this study were classified according to the American College of Medical Genetics and Genomics (ACMG) guidelines.

Total genomic DNA was extracted following the protocol described by Song et al. (2006) (link). Twelve microsatellites were selected from 100 Asian cultivated rice microsatellites (www.gramene.org) and 16 Z. latifolia specific microsatellites (Quan et al., 2009 (link)) (Table S1). The PCR products were labeled with fluorescent dyes using 5′-tagged forward primers (FAM, JOE, and ROX) and GS350 as an internal size standard labeled with TAMRA. The PCR products were sequenced on an ABI 3730 (Applied Biosystems) automated sequencer. The resulting chromatograms were visualized and analyzed using GENEMAPPER v4.0 software (Applied Biosystems).
The ITS region of smut fungus was amplified using ITS1 and ITS4. PCR products were purified and sequenced on an ABI 3130XL capillary sequencer (Applied Biosystems). All ITS sequences were deposited into GeneBank, with accession numbers MK811211-MK811247.

Genotyping relied on the analysis of eight polymorphic microsatellite markers (Poly α, TA109, TA1, TA81, TA42, ARA2, PfPK2, Pfg377) distributed among five chromosomes of P. falciparum, as previously published [20 (link), 29 (link)]. Microsatellites were amplified via a two-step, semi-nested PCR using fluorescent end-labelled primers as previously described [29 (link), 30 (link)]. Primer sequences, chromosome location, as well as reaction and cycling conditions are detailed in Additional file 1. To determine repeat-length sizes, PCR products were analysed using an ABI 3130xl capillary sequencer (Applied Biosystems, Foster City, CA, USA). Microsatellite allele length was determined using internal size standards (GeneScan 500 LIZ Size Standard, Applied Biosystems) with the GeneMapper v4.0 software (Applied Biosystems). To differentiate allele peaks from stutter peak artefacts, multiple alleles per locus were scored if minor peaks were >33 % of the height of the major peak, corresponding to the predominant allele [20 (link), 29 (link)]. Peaks with a fluorescence intensity <100 units were discarded.