3730xl sequencer

Manufactured by Thermo Fisher Scientific

Sourced in United States, France

The 3730XL sequencer is a high-throughput genetic analysis instrument designed for DNA sequencing. It utilizes capillary electrophoresis technology to analyze DNA samples and generate sequencing data. The 3730XL is capable of processing multiple samples simultaneously, providing efficient and reliable genetic information for research and diagnostic applications.

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Lab products found in correlation

Plant genomic dna kit, by Tiangen Biotech (6 mentions) Qiaquick gel extraction kit, by Qiagen (3 mentions) Abi prism bigdye terminator cycle sequencing kit, by Thermo Fisher Scientific (2 mentions) Qiaamp blood kit, by Qiagen (2 mentions) Accuprime pfx dna polymerase, by Thermo Fisher Scientific (2 mentions) Qiaquick pcr purification kit, by Qiagen (2 mentions) Nextera rapid capture exome kit, by Illumina (1 mentions) Sureselect human all exon kit, by Agilent Technologies (1 mentions) Hiseq 4000, by Illumina (1 mentions) Spss statistics for windows version 26, by IBM (1 mentions) Seqscape software, by Thermo Fisher Scientific (1 mentions) Bigdye v3, by Thermo Fisher Scientific (1 mentions) Gotaq g2 hot start master mix, by Promega (1 mentions) Qiaquick pcr purification spin columns, by Qiagen (1 mentions) Casava v1.8 pipeline, by Illumina (1 mentions) Multifunction dna purification kit, by BioTeke (1 mentions) Biotek epoch, by Agilent Technologies (1 mentions) Alamut visual, by Sophia Genetics (1 mentions) Bigdye terminator 3.1 kit, by Thermo Fisher Scientific (1 mentions) Platinum hot start pcr master mix, by Thermo Fisher Scientific (1 mentions)

64 protocols using 3730xl sequencer

Whole Exome Sequencing for Genetic Diagnosis

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Patient blood samples were referred for routine genetic diagnostic workup by experienced clinical geneticists. DNA was extracted by standard methods. WES was performed as described previously [7 (link)]. In short, the Nextera Rapid Capture Exome Kit (Illumina, San Diego, CA) or the SureSelect Human All Exon kit (Agilent, Santa Clara, CA) were used for enrichment, and a HiSeq4000 (Illumina) instrument for the actual sequencing with the average coverage targeted to 100×. Variants calling, annotation and prioritization was based on a set of publicly available and in house developed tools. WES was performed in index, parents and affected nephew from family 1 (I-1, I-2, II-8, III-1), index case from family 2 (II-2) and index case and parents from family 3.
The variant-containing exons of CXorf56 (NM_022101.3) were amplified (primers available upon request) and Sanger-sequenced from both sides on a 3730xl sequencer (Thermo Fisher Scientific, Waltham, MA) in all available family members for co-segregation analysis. Exon numbering was used according to reference sequence NM_022101.3 (exons 1–7).
Detected CXorf56 variants have been submitted to the Leiden Open Variation database, http://www.lovd.nl/CXorf56 (Individual IDs: 00266162 and 00266164).

Rocha M.E., Silveira T.R., Sasaki E., Sás D.M., Lourenço C.M., Kandaswamy K.K., Beetz C., Rolfs A., Bauer P., Reardon W, & Bertoli-Avella A.M. (2019). Novel clinical and genetic insight into CXorf56-associated intellectual disability. European Journal of Human Genetics, 28(3), 367-372.

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Amplification and Sequencing of ANTXR2

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The coding sequence of ANTXR2 (NM_058172.5; NP_477520.2) including at least 50 bp of adjacent untranslated regions or intronic sequences was amplified exon-wise from genomic DNA (primers available upon request). PCR-products were extracted from agarose gels, purified according to standard procedures, and sequenced from both sides on a 3730xl sequencer (Thermo Fisher Scientific, Waltham, MA).

Cozma C., Hovakimyan M., Iurașcu M.I., Makhseed N., Selim L.A., Alhashem A.M., Ben-Omran T., Mahmoud I.G., Al Menabawy N.M., Al-Mureikhi M., Martin M., Demuth L., Yüksel Z., Beetz C., Bauer P, & Rolfs A. (2019). Genetic, clinical and biochemical characterization of a large cohort of patients with hyaline fibromatosis syndrome. Orphanet Journal of Rare Diseases, 14, 209.

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Antibiotic Resistance Profiling Protocol

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Clade PA, USA) and sent to Macrogen Inc. (Seoul, Korea) for Sanger sequencing on an Applied Biosystems 3730xl sequencer (Thermo Fisher Scientific, Waltham, MA, USA). The nucleotide and amino acid sequences identified in this study were aligned to sequences in the Beta-Lactamase DataBase (http://bldb.eu/) (10) . Data and statistical analyses: Data were analyzed using GraphPad Prism, version 8.0.1 (GraphPad Software Inc., San Diego, CA, USA). Statistical analysis was performed using IBM SPSS Statistics for Windows, version 26.0 (IBM Corp., Armonk, NY, USA). Welch's t-test or independent t-test with unequal variances was used for mean comparisons between pairs of independent groups. Statistical significance was considered when the probability (P) value was less than 0.05.
Ethical considerations: This nonclinical study was approved by the Institutional Review Board of Siriraj Hospital (approval numbers: Si479/2015 and Si720/2018).

Nokchan N., Nitayanon P, & Tribuddharat C. (2023). Molecular Epidemiology of Penicillinase-Producing Neisseria gonorrhoeae Isolates and Their bla_TEM-135 Gene Variant in Bangkok, Thailand, 2015-2017. Japanese journal of infectious diseases, 76(2).

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Sanger Sequencing for Mutation Segregation

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The intrafamilial segregation of pathogenic mutations was performed by Sanger sequencing (BigDye v3.1, Life Technologies) of the PCR product obtained with the GoTaq ® G2 Hot Start Master Mix (Promega) and purified with ExoSAP (UBS). Sequences were analyzed on a 3730XL sequencer and with the SeqScape software (Life Technologies). Purified PCR products were sequenced using forward and reverse amplification primers (primer sequences are available upon request).

Mabrouk I., Al-Harthi N., Mani R., Montantin G., Tissier S., Lagha R., Ben Abdallah F., Hassan M.M., Alhomrani M., Gaber A., Alsanie W.F., Ouali H., Jambi F.A., Almaghamsi T.M., Alqarni N.A., Alfarsi N.A., Kashgari K., Al-Zahrani H.J., Al-Shamary Z.A., Al-Harbi A., Amselem S., Escudier E, & Legendre M. (2022). Combining RSPH9 founder mutation screening and next-generation sequencing analysis is efficient for primary ciliary dyskinesia diagnosis in Saudi patients. Journal of human genetics, 67(7).

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Ribosomal ITS Sequencing of Yeast Strains

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The ribosomal internal transcribed spacer (ITS) sequences were retrieved from GenBank (NCBI database) under the accession num bers indicated in Table 1. Where sequences were not available in the database, analysis was performed according to James et al. (2014) . Yeast cells were breaked using microwaves (Panasonic, 800 W) for 30 s in 50 lL of water to obtain cells extracts. The ITS region was amplified by PCR directly from whole yeast cell extracts, amplified using primers ITS5 and ITS4 and sequenced using these primers as well as internal primers ITS2 and ITS3. The amplified DNA was checked by 1% agarose gel electrophoresis, purified and concentrated using QIAquick PCR purification spin columns (Qiagen) and sequenced using a Life Technologies 3730XL sequencer at the Genome Analysis Centre (TGAC), Nor wich, UK. Pairwise alignments and phylogenetic analysis were con ducted using Geneious 7.1 software created by Biomatters. A phylogenetic neighbour joining tree was then generated using the distance Tamura Nei model. Confidence values for branch nodes were estimated from bootstrap analyses of 1000 replicates Yeast producing strains (Cardinali et al., 2012; Urubschurov, Janczyk, Souffrant, Freyer, & Zeyner, 2011) .

Grondin E., Shum Cheong Sing A., James S., Nueno-Palop C., François J.M, & Petit T. (2017). Flavour production by Saprochaete and Geotrichum yeasts and their close relatives. Food chemistry, 237.

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Identifying Pathogenic Variants via NGS

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Sequencing reads were generated by the Illumina CASAVA v1.8 pipeline and aligned to the human reference genome (hg19) using the Burrows–Wheeler Aligner (BWA) program. Variants were called using the GATK package v4.1.8.1. All variants were annotated and characterized using ANNOVAR software. To identify pathogenic variants, we filtered out the following: (1) low-quality variants (depth < 10, or genotype quality < 30); (2) variants in the noncoding regions, except for those that might disrupt splicing; (3) synonymous variants in the coding region; (4) variants with minor allele frequency (MAF) > 0.001 in several databases (1000 Genome Project, gnomad v2.1.1 and in-house database); and (5) variants labeled as “benign” in the ClinVar database. The deleterious effect of variants was predicted by SIFT scores, REVEL, and CADD scores. To validate the variants, Sanger sequencing of PDZD7 exon 15 was performed on genomic DNA from all family members and 96 normal hearing controls. PCR and sequencing primers were designed by Primer3 online software. Sanger sequencing was performed on a 3730XL sequencer (Applied Biosystems) according to the manufacturer’s instructions.

Du Q., Sun Q., Gu X., Wang J., Li W., Guo L, & Li H. (2022). Novel homozygous variant in the PDZD7 gene in a family with nonsyndromic sensorineural hearing loss. BMC Medical Genomics, 15, 135.

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Plant DNA Extraction and Barcoding Procedure

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For each sample, 30–40 mg of leaves dried by silica gel were used, and genomic DNA was extracted and purified according to the Plant Genomic DNA Kit (Tiangen Biotech Co., China). The DNA concentration was estimated using BioTek Epoch (BioTek, Co., United States) by standard spectrophotometric methods at 260 and 280 nm. DNA integrity was assessed by electrophoresis using 1.0% agarose gel. Then, the DNA samples were diluted to a working concentration of 50 ng/μl and stored at −20°C until further use. According to Zhang et al. (2012) (link), four commonly used DNA barcoding loci (matK, rbcL, psbA-trnH, and ITS) were used in this study. The steps were carried out according to the China Plant BOL Group (2011) (link) and Chen et al. (2010) (link) using DNA barcoding standard operating procedures (DNA barcoding SOP).
PCR amplification was performed in 25 μl reaction mixtures containing 20–50 ng of genomic DNA, 12.5 μl of 2 × Taq PCR MasterMix (Beijing Aidlab Biotech Co., Beijing, China), 1 μl of 2.5 μM forward and reverse primers, and distilled water up to the final volume. PCR products were assessed on 1.0% agarose gel, visualized under UV light, purified using a Multifunction DNA Purification Kit from Bioteke (China) and then sequenced in both directions on a 3730XL sequencer (Applied Biosystems, United States) using amplification primers listed in Supplementary Table 2 (Chen, 2015 ).

Xiong C., Sun W., Wu L., Xu R., Zhang Y., Zhu W., J. H.E., P.a.n.j.w.a.n.i., Liu Z, & Zhao B. (2022). Evaluation of Four Commonly Used DNA Barcoding Loci for Ardisia Species Identification. Frontiers in Plant Science, 13, 860778.

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Amplification and Sequencing of ApoE3ch Variant

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Reaction mixture for the amplification process was performed in a 50 μL volume that included the following components: 1xPfuUltra II Hostart Master Mix, 1μL of each primer (10 μmol/L)(Forward primer: 5’- AGCCCTTCTCCCCGCCTCCCACTGT-3’ and Reverse primer: 5’- CTCC GCCACCTGCTCCTTCACCTCG-3’), 5% DMSO and 1μL of genomic DNA (100 ng/μL)^{21 (link)}. PCR cycling was run with initial denaturation at 95°C for 2 min followed by 35 cycles with denaturation at 95°C for 20 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 40 seconds, and a final extension at 72°C for 5 minutes. PCR products were purified using QIAquick Gel Extraction kit from Qiagen and sequenced by MGH CCIB DNA core using the 3730xl sequencer from Applied Biosystems. To avoid errors in sampling the blood sample used for Sanger sequencing was different from the sample used for the WGS analysis. Genotyping of four descendants of the case were confirmed to be heterozygote carriers of the ApoE3ch as expected when one parent is homozygote (Extended data 2). Data is representative of n = 2 independent experiments and further validated independently with whole exome and whole genome sequencing. Further information can be found in the Life Sciences Reporting Summary.

Arboleda-Velasquez J.F., Lopera F., O’Hare M., Delgado-Tirado S., Marino C., Chmielewska N., Saez-Torres K.L., Amarnani D., Schultz A.P., Sperling R.A., Leyton-Cifuentes D., Chen K., Baena A., Aguillon D., Rios-Romenets S., Giraldo M., Guzmán-Vélez E., Norton D.J., Pardilla-Delgado E., Artola A., Sanchez J.S., Acosta-Uribe J., Lalli M., Kosik K.S., Huentelman M.J., Zetterberg H., Blennow K., Reiman R.A., Luo J., Chen Y., Thiyyagura P., Su Y., Jun G.R., Naymik M., Gai X., Bootwalla M., Ji J., Shen L., Miller J.B., Kim L.A., Tariot P.N., Johnson K.A., Reiman E.M, & Quiroz Y.T. (2019). Resistance to autosomal dominant Alzheimer’s in an APOE3-Christchurch homozygote: a case report. Nature medicine, 25(11), 1680-1683.

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Plant DNA Extraction and Sequencing

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All samples (40 mg) were rubbed for 2 min at a frequency of 30 r/s. Total genomic DNA was extracted using the Plant Genomic DNA Kit (Tiangen Biotech Co.) according to the manufacturer's instructions. The extracted genomic DNA was amplified by polymerase chain reaction (PCR) using the ITS (ITS5F and ITS4R) and psbA‐trnH (fwdPA and revTH) primers (Chen, 2012). The PCR mixtures and conditions were described by Chen (2012). PCR products were separated and detected by 1.5% agarose gel electrophoresis. Purified products were sequenced in both directions using the PCR primers on a 3730XL sequencer (Applied Biosystems).

Zhang Z., Wang X., Zhang Z., Yao H., Zhang X., Zhang Y, & Zhang B. (2019). The impact of genetic diversity on the accuracy of DNA barcoding to identify species: A study on the genus Phellodendron. Ecology and Evolution, 9(18), 10723-10733.

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Genetic Profiling of ABCC6 Gene Variants

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Genomic DNA was isolated from whole blood (QIAamp blood kit,
Qiagen^®, Hilden, Germany) according to an established
protocol. The complete ABCC6 coding region was amplified using
previously described PCR primers (51 (link)).
For the detection of the multi-exon 23–29 deletion, primers were used as
described by Le Saux et al (31 (link)).
The ABCC6 coding region and intron/exon boundaries were
analyzed through direct sequencing using an Applied Biosystems 3730xl
Sequencer^®, with ABI PRISM BigDye Terminator Cycle
Sequencing Kit (Applied Biosystems^®. Foster City, USA). For
variant classification the gnomAD, and Alamut ^® Visual
(Interactive Biosoftware, Rouen, France) were used (14 (link),32 ). To
assess conservation of the variants, multiple sequence alignment was performed
for the following species: Homo sapiens, Pan troglodytes, Mus musculus, Rattus
norvegicus, and Danio rerio using the Clustal Omega software (46 ). Unreported sequence variants were defined as
pathogenic based on criteria reported by the American College of Medical
Genetics and Genomics and the Association for Molecular Pathology, taken into
account that we assessed a complex phenotype, which hinders the determination of
the specificity of the phenotype to the variant and complicates segregation
analysis if not clear (43 (link)). Nucleotide
numbers are derived from gDNA ABCC6 sequences (GenBank
accession no. NM_001171).

De Vilder E.Y., Cardoen S., Hosen M.J., Le Saux O., De Zaeytijd J., Leroy B.P., De Reuck J., Coucke P.J., De Paepe A., Hemelsoet D, & Vanakker O.M. (2018). Pathogenic variants in the ABCC6 gene are associated with an increased risk for ischemic stroke. Brain Pathology, 28(6), 822-831.

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