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Challenges in the introduction of next-generation sequencing (NGS) for diagnostics of myeloid malignancies into clinical routine use.
Given the huge phenotypic and genetic heterogeneity of acute and continual myeloid malignancies, hematologists have eagerly awaited the introduction of next-generation sequencing (NGS) into the routine diagnostic armamentarium to allow a extra differentiated illness classification, threat stratification, and improved therapeutic choices. At current, an rising variety of hematologic laboratories are within the technique of integrating NGS procedures into the diagnostic algorithms of sufferers with acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and myeloproliferative neoplasms (MPNs).
Inevitably accompanying such developments, physicians and molecular biologists are dealing with sudden challenges concerning the interpretation and implementation of molecular genetic outcomes derived from NGS in myeloid malignancies. This text summarizes typical challenges (Desk 1) which will come up within the context of NGS-based analyses at analysis and through follow-up of myeloid malignancies.
Desk 1 Challenges accompanying the introduction of large parallel sequencing in scientific routine diagnostics in hemato-oncology Problem Background Present and future method Discrimination of leukemia-related mutations from polymorphisms or passenger mutations Driver mutations anticipated to happen at increased allele frequency in affected person samples than passenger mutations; driver mutations extra prone to have an effect on protein perform than polymorphisms or passenger mutations Optimization of cancer-specific databases together with reporting of uncommon physiological gene variants Implementation of novel bioinformatic algorithms primarily based on prediction of useful influence Quantitative and dynamic.
VAF monitoring (individually and along with different mutations) at follow-up Discrimination of somatic leukemia-related mutations from CHIP CHIP is offered in ~10% of people aged 70 to 80 and in as much as 20% within the age group>> 80 years Quantitative and dynamic VAF monitoring (individually and along with different mutations) at follow-up Clarifying the importance of CHIP within the context of myeloid malignancies.
Discrimination of leukemia-related somatic mutations from pathogenic germline alterations Problem to distinguish acquired somatic mutations from germline pathogenic variants at analysis Mutation detection in germline management samples (e.g., pores and skin fibroblasts, saliva) in mutations corresponding to in RUNX1, CEBPA Thorough medical household historical past adopted by molecular genetic checks in kinfolk if essential Excessive and secure VAF (e.g., 40-50%) at follow-up regardless of scientific response to therapy could also be indicative for germline alteration.
Discrimination of true genetic alterations from PCR, sequencing and post-sequencing artifacts Many artefacts are identified to come up throughout NGS library preparation, sequencing and information evaluation Error correction utilizing molecular identifiers that individually label unique enter DNA molecules Refinement of error-correction computational strategies in post-sequencing NGS information evaluation Affirmation utilizing Sanger sequencing Restricted sensitivity of NGS for minimal residual illness (MRD) evaluation Mutations detected at analysis could also be re-identified at finest to a VAF of 1-2% Error-corrected sequencing utilizing molecular identifiers.
Complementation of NGS by established MRD instruments like real-time PCR and movement cytometry Excessive monetary burden; demand on interdisciplinary approaches Costly technical and workers gear, refined information interpretation Advanced translation of NGS outcomes into therapeutic choices Growth of repeatedly up to date NGS interpretation units and algorithms for well-established mutational profiles inside distinct hematological malignancies Interdisciplinary leukemia boards VAF variant allele frequency, CHIP clonal hematopoiesis of indeterminate significance, bp base pairs, G guanine, C cytosine, ITDs inside tandem duplication.
NGS-QCbox and Raspberry for Parallel, Automated and Fast High quality Management Evaluation of Massive-Scale Subsequent Technology Sequencing (Illumina) Knowledge.
Fast reputation and adaptation of subsequent technology sequencing (NGS) approaches have generated large volumes of knowledge. Excessive throughput platforms like Illumina HiSeq produce terabytes of uncooked information that requires fast processing. High quality management of the information is a crucial element previous to the downstream analyses. To deal with these points, we have now developed a high quality management pipeline, NGS-QCbox that scales as much as course of lots of or 1000’s of samples. Raspberry is an in-house device, developed in C language using HTSlib (v1.2.1) (http://htslib.org), for computing learn/base degree statistics.
It may be used as stand-alone utility and may course of each compressed and uncompressed FASTQ format recordsdata. NGS-QCbox integrates Raspberry with different open-source instruments for alignment (Bowtie2), SNP calling (SAMtools) and different utilities (bedtools) in the direction of analyzing uncooked NGS information at increased effectivity and in high-throughput method.
It stories learn and base statistics together with genome protection and variants in a consumer pleasant format. The pipeline developed presents a easy menu pushed interface and can be utilized in both fast or full mode. As well as, the pipeline in fast mode outperforms in pace in opposition to different related present QC pipeline/instruments.
Diminishing returns in next-generation sequencing (NGS) transcriptome information.
RNA-seq is more and more used to review gene expression of assorted organisms. Whereas it supplies an ideal alternative to discover genome-scale transcriptional patterns with large depth, it comes with prohibitive prices. Establishing a minimal sequencing depth for required accuracy will information cost-effective experimental design and promote the routine utility of RNA-seq. To deal with this subject, we chosen 36 RNA-seq datasets, every with greater than 20 million reads from six widely-used mannequin organisms: Saccharomyces cerevisiae, Homo sapiens, Drosophila melanogaster, Caenorhabditis elegans, Mus musculus, and Arabidopsis thaliana, and investigated statistical correlations between the sequencing depth and the result accuracy.
) Disulfide Oxidoreductase (Recombinant) |
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| 20-abx073036 | Abbexa |
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 Formamidine disulfide dihydrochloride |
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| GK5373-25G | Glentham Life Sciences | 25 g | EUR 134 |
 Recombinant Disulfide Bond Isomerase |
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| 7-02695 | CHI Scientific | 10µg | Ask for price |
Recombinant Disulfide Bond Isomerase |
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| 7-02696 | CHI Scientific | 50µg | Ask for price |
Recombinant Disulfide Bond Isomerase |
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| 7-02697 | CHI Scientific | 1mg | Ask for price |
 Protein disulfide-isomerase Antibody |
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| 20-abx110136 | Abbexa |
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Protein disulfide-isomerase Antibody |
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| 20-abx110137 | Abbexa |
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) Disulfide Isomerase Protein (Recombinant) |
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| 20-abx073027 | Abbexa |
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) Disulfide Bond Isomerase (Recombinant) |
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| 20-abx073037 | Abbexa |
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 N1-Glutathionyl-spermidine disulfide |
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| H-1126.0005 | Bachem | 5.0mg | EUR 454 |
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Description: Sum Formula: C34H66N12O10S2; CAS# [108081-77-2] |
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N1-Glutathionyl-spermidine disulfide |
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| H-1126.0025 | Bachem | 25.0mg | EUR 1723 |
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Description: Sum Formula: C34H66N12O10S2; CAS# [108081-77-2] |
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 Disulfide Oxidoreductase) Recombinant (E.Coli) Disulfide Oxidoreductase |
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| RP-365 | Alpha Diagnostics | 10 ug | EUR 225 |
 Recombinant Human Protein Disulfide Isomerase |
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| 7-03304 | CHI Scientific | 100µg | Ask for price |
Recombinant Human Protein Disulfide Isomerase |
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| 7-03305 | CHI Scientific | 500µg | Ask for price |
Recombinant Human Protein Disulfide Isomerase |
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| 7-03306 | CHI Scientific | 1mg | Ask for price |
 Protein disulfide-isomerase Polyclonal Antibody |
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| 42079-100ul | SAB | 100ul | EUR 333 |
 Antibody) PDI (Protein Disulfide Isomerase) Antibody |
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| 5601-100 | Biovision | EUR 316 | |
PDI (Protein Disulfide Isomerase) Antibody |
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| 5601-30T | Biovision | EUR 146 | |
) Human Protein disulfide-isomerase (P4HB) |
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| 1-CSB-EP017342HU | Cusabio |
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Description: Recombinant Human Protein disulfide-isomerase(P4HB) expressed in E.coli |
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Human Protein disulfide-isomerase (P4HB) |
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| 1-CSB-RP002544h | Cusabio |
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Description: Recombinant Human Protein disulfide-isomerase(P4HB),partial expressed in E.coli |
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) Human Protein disulfide-isomerase (P4hb) |
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| 1-CSB-YP017342HU | Cusabio |
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Description: Recombinant Human Protein disulfide-isomerase(P4hb) expressed in Yeast |
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Human Protein disulfide-isomerase (P4HB) |
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| 1-CSB-YP017342HUf8 | Cusabio |
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Description: Recombinant Human Protein disulfide-isomerase(P4HB),partial expressed in Yeast |
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 Antibody) Protein Disulfide Isomerase (PDI) Antibody |
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| 20-abx102689 | Abbexa |
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Protein Disulfide Isomerase (PDI) Antibody |
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| 20-abx102690 | Abbexa |
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) Protein disulfide-isomerase Antibody (Biotin) |
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| 20-abx105821 | Abbexa |
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Protein disulfide-isomerase Antibody (Biotin) |
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| 20-abx105825 | Abbexa |
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) Protein disulfide-isomerase Antibody (FITC) |
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| 20-abx107238 | Abbexa |
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Protein disulfide-isomerase Antibody (FITC) |
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| 20-abx107239 | Abbexa |
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) Protein disulfide-isomerase Antibody (HRP) |
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| 20-abx108658 | Abbexa |
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Protein disulfide-isomerase Antibody (HRP) |
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| 20-abx108659 | Abbexa |
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 Protein disulfide-isomerase TMX3 Antibody |
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| 20-abx110139 | Abbexa |
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 Antibody) Protein Disulfide-Isomerase (P4HB) Antibody |
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| 20-abx126992 | Abbexa |
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Protein Disulfide Isomerase (PDI) Antibody |
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| 20-abx128556 | Abbexa |
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Protein Disulfide-Isomerase (P4HB) Antibody |
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| abx145769-100ug | Abbexa | 100 ug | EUR 391 |
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Protein Disulfide Isomerase (PDI) Antibody |
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| 20-abx141570 | Abbexa |
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Protein Disulfide Isomerase (PDI) Antibody |
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| 20-abx174245 | Abbexa |
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) Disulfide Isomerase A3 Protein (Recombinant) |
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| 20-abx073097 | Abbexa |
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) Disulfide Isomerase A6 Protein (Recombinant) |
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| 20-abx073169 | Abbexa |
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) Disulfide Isomerase A4 Protein (Recombinant) |
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| 20-abx073751 | Abbexa |
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Protein Disulfide-Isomerase (P4HB) Antibody |
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| abx031784-400ul | Abbexa | 400 ul | EUR 523 |
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Protein Disulfide-Isomerase (P4HB) Antibody |
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| abx031784-80l | Abbexa | 80 µl | EUR 286 |
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Protein Disulfide Isomerase (PDI) Antibody |
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| abx236272-100ug | Abbexa | 100 ug | EUR 481 |
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 Antibody) Protein Disulfide Isomerase (P4HB) Antibody |
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| 20-abx445165 | Abbexa |
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Protein Disulfide-Isomerase (P4HB) Antibody |
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| 20-abx300560 | Abbexa |
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Protein Disulfide-Isomerase (P4HB) Antibody |
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| 20-abx000827 | Abbexa |
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 PDI, Protein Disulfide Isomerase, human |
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| RC512-13 | Bio Basic | 100ug | EUR 256.63 |
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 DsbA Disulfide Oxidoreductase Recombinant Protein |
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| PROTP0AEG4 | BosterBio | Regular: 50ug | EUR 317 |
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Description: Disulfide Oxidoreductase produced in E.Coli is a periplasmic protein isolated from E. coli, containing 208 amino acids having a molecular mass of 23,149 Dalton. ;The DsbA is purified by proprietary chromatographic techniques. |
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) Recombinant Protein Disulfide Isomerase (PDI) |
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| 4-RPB061Hu01 | Cloud-Clone |
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Description: Recombinant Human Protein Disulfide Isomerase expressed in: E.coli |
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Recombinant Protein Disulfide Isomerase (PDI) |
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| 4-RPB061Mu01 | Cloud-Clone |
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Description: Recombinant Mouse Protein Disulfide Isomerase expressed in: E.coli |
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Recombinant Protein Disulfide Isomerase (PDI) |
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| 4-RPB061Ra01 | Cloud-Clone |
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Description: Recombinant Rat Protein Disulfide Isomerase expressed in: E.coli |
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 Disulfide Bond Isomerase) Recombinant (E.Coli) Disulfide Bond Isomerase |
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| RP-378 | Alpha Diagnostics | 10 ug | EUR 225 |
 Recombinant E.Coli Thiol Disulfide Interchange Protein |
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| 7-06964 | CHI Scientific | 5µg | Ask for price |
Recombinant E.Coli Thiol Disulfide Interchange Protein |
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| 7-06965 | CHI Scientific | 25µg | Ask for price |
Recombinant E.Coli Thiol Disulfide Interchange Protein |
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| 7-06966 | CHI Scientific | 1mg | Ask for price |
Recombinant E.Coli Thiol Disulfide Interchange Protein |
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| 7-06967 | CHI Scientific | 5µg | Ask for price |
Recombinant E.Coli Thiol Disulfide Interchange Protein |
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| 7-06968 | CHI Scientific | 25µg | Ask for price |
Recombinant E.Coli Thiol Disulfide Interchange Protein |
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| 7-06969 | CHI Scientific | 1mg | Ask for price |
 Recombinant Human Protein Disulfide Isomerase A3 |
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| 7-03307 | CHI Scientific | 5µg | Ask for price |
Recombinant Human Protein Disulfide Isomerase A3 |
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| 7-03308 | CHI Scientific | 25µg | Ask for price |
Recombinant Human Protein Disulfide Isomerase A3 |
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| 7-03309 | CHI Scientific | 1mg | Ask for price |
 Recombinant Human Protein Disulfide Isomerase A4 |
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| 7-03310 | CHI Scientific | 2µg | Ask for price |
Recombinant Human Protein Disulfide Isomerase A4 |
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| 7-03311 | CHI Scientific | 10µg | Ask for price |
Recombinant Human Protein Disulfide Isomerase A4 |
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| 7-03312 | CHI Scientific | 1mg | Ask for price |
 Recombinant Human Protein Disulfide Isomerase A6 |
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| 7-03313 | CHI Scientific | 5µg | Ask for price |
Recombinant Human Protein Disulfide Isomerase A6 |
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| 7-03314 | CHI Scientific | 20µg | Ask for price |
Recombinant Human Protein Disulfide Isomerase A6 |
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| 7-03315 | CHI Scientific | 1mg | Ask for price |
) Mouse Protein disulfide-isomerase A2 (Pdia2) |
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| 1-CSB-EP017719MO | Cusabio |
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Description: Recombinant Mouse Protein disulfide-isomerase A2(Pdia2) expressed in E.coli |
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) Human Protein disulfide-isomerase A3 (PDIA3) |
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| 1-CSB-EP017720HU | Cusabio |
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Description: Recombinant Human Protein disulfide-isomerase A3(PDIA3) expressed in E.coli |
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) Cricetulus griseus Protein disulfide-isomerase (P4HB) |
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| 1-CSB-EP823188DXU | Cusabio |
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Description: Recombinant Cricetulus griseus Protein disulfide-isomerase(P4HB) expressed in E.coli |
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) Human Protein disulfide-isomerase TMX3 (TMX3) |
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| 1-CSB-EP846645HU | Cusabio |
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Description: Recombinant Human Protein disulfide-isomerase TMX3(TMX3) expressed in E.coli |
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Mouse Protein disulfide-isomerase A2 (Pdia2) |
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| 1-CSB-YP017719MO | Cusabio |
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Description: Recombinant Mouse Protein disulfide-isomerase A2(Pdia2) expressed in Yeast |
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Cricetulus griseus Protein disulfide-isomerase (P4HB) |
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| 1-CSB-YP823188DXU | Cusabio |
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Description: Recombinant Cricetulus griseus Protein disulfide-isomerase(P4HB) expressed in Yeast |
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, human recombinant) Protein Disulfide Isomerase (PDI), human recombinant |
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| 7601-100 | Biovision | EUR 262 | |
Protein Disulfide Isomerase (PDI), human recombinant |
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| 7601-1000 | Biovision | EUR 1246 | |
Protein Disulfide Isomerase (PDI), human recombinant |
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| 7601-5000 | Biovision | EUR 4345 | |
 Antibody) Protein Disulfide Isomerase A6 (PDIA6) Antibody |
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| 20-abx005339 | Abbexa |
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 Antibody) Protein Disulfide Isomerase A5 (PDIA5) Antibody |
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| abx027813-400ul | Abbexa | 400 ul | EUR 523 |
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Protein Disulfide Isomerase A5 (PDIA5) Antibody |
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| abx027813-80l | Abbexa | 80 µl | EUR 286 |
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 Antibody) Protein Disulfide-Isomerase A4 (PDIA4) Antibody |
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| abx027892-400ul | Abbexa | 400 ul | EUR 523 |
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Protein Disulfide-Isomerase A4 (PDIA4) Antibody |
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| abx027892-80l | Abbexa | 80 µl | EUR 286 |
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Protein Disulfide-Isomerase A4 (PDIA4) Antibody |
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| 20-abx003217 | Abbexa |
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 Antibody) Protein Disulfide Isomerase A2 (PDIA2) Antibody |
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| 20-abx178145 | Abbexa |
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 Antibody) Protein Disulfide Isomerase A4 (PDIA4) Antibody |
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| 20-abx178146 | Abbexa |
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Protein Disulfide Isomerase A4 (PDIA4) Antibody |
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| 20-abx178147 | Abbexa |
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Protein Disulfide Isomerase A2 (PDIA2) Antibody |
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| 20-abx210340 | Abbexa |
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Protein Disulfide Isomerase A2 (PDIA2) Antibody |
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| 20-abx210341 | Abbexa |
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Protein Disulfide Isomerase A2 (PDIA2) Antibody |
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| 20-abx103307 | Abbexa |
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 Antibody) Protein Disulfide Isomerase A3 (PDIA3) Antibody |
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| 20-abx103308 | Abbexa |
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) Protein disulfide-isomerase A2 Antibody (Biotin) |
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| 20-abx105822 | Abbexa |
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) Protein disulfide-isomerase A3 Antibody (Biotin) |
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| 20-abx105823 | Abbexa |
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) Protein disulfide-isomerase TMX3 Antibody (Biotin) |
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| 20-abx105824 | Abbexa |
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) Protein disulfide-isomerase A2 Antibody (FITC) |
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| 20-abx107240 | Abbexa |
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) Protein disulfide-isomerase A3 Antibody (FITC) |
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| 20-abx107241 | Abbexa |
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) Protein disulfide-isomerase TMX3 Antibody (FITC) |
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| 20-abx107242 | Abbexa |
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) Protein disulfide-isomerase A2 Antibody (HRP) |
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| 20-abx108660 | Abbexa |
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) Protein disulfide-isomerase A3 Antibody (HRP) |
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| 20-abx108661 | Abbexa |
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To realize this, we randomly selected reads from every dataset, mapped them to the reference genomes, and analyzed the accuracy achieved with various protection. Our outcomes indicated that as little as a million reads can present the identical sequencing accuracy in transcript abundance (r=0.99) as >30 million reads for highly-expressed genes in all six species. As a result of many metabolically and pathologically-relevant genes are extremely expressed, our findings is perhaps instructive for cost-effective experimental designs in NGS-based analysis and likewise present helpful steerage to related analysis for different organisms.
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