2. 9. 4, Broad Institute, Cambridge, MA). A hotspots bed file was loaded to facilitate the mutation findings with the more frequent mutations reported. Digital PCRcDNA samples were analyzed using a commercially available custom TaqManВ® assay on a QuantStudioВ® 3D Digital PCR System (Thermo Fisher, Palo Alto, CA) to confirm p. T315I mutation with TaqMan Assay probe/primer set: C_174580870_10 (Thermo Fisher Scientific, Palo Alto, CA), including FAM-labeled probe for p.
Figure 1Study flow chart. Overview of the design and distribution of the patients and samples analyzed by RNA-NestedNGS. ALL acute lymphoblastic leukemia, CML chronic myeloid leukemia, D diagnosis, ELN European leukemia net, F failure, R relapse, W warning. Figure 2(A) Scheme methodology of the detection of BCR::ABL1 mutations by RNA-NestedNGS; (B) Correlation between the two replicates by RNA-NestedNGS; (C) Histogram of mutational frequency by RNA-NestedNGS method in failure/warning stages for CML patients and diagnosis/relapse ALL patients; (D) Results from EUTOS international control round for deep sequencing analysis of BCR::ABL1 mutations using RNA-NestedNGS approach showing the ability of the nested method to detect KD mutations. ALL acute lymphoblastic leukemia, CML chronic myeloid leukemia, KD kinase domain.
This test was validated by the EUTOS ring trial for BCR::ABL1 mutations where 91% of the samples (20/22) were correctly reported in both mutation and allelic frequency (Fig. 2D). Most of the mutations observed were in failure and relapse stages for CML and B-ALL patients, respectively, as previously reported11, 19. Our DNA-DeepNGS method was able to detect mutations with a strong reproducibility using a threshold of VAF of 1. 0Eв€’4, based on the comparison with RNA-NestedNGS in a ROC analysis (Fig.
Ismael Gonzalez: Real Murcia part ways with Kenya midfielder Real Murcia. African All StarsPrimera División RFEFTransfersUCAM MurciaI.
AthumanKenya. The 27-year-old Harambee
The BCR::ABL1 levels in B-ALL patients were not corrected for the international standardization factor. The main clinical characteristics are included in Table 1. The study was conducted in accordance with the principles of the Declaration of Helsinki, and the project has been reported as favorable by the Ethics Committee of the Hospital 12 de Octubre (CEI number: 16/187). All patients provided written informed consent for the analysis of their biological specimens. Table 1 Summary of the main data of the two cohorts. Sanger SequencingSS screening of the BCR::ABL1 KD was carried out on an Applied BiosystemsВ® 3130 Genetic Analyzer (Thermo Fisher, Palo Alto, CA) as follows: RNA was extracted from fresh BM/PB using a standard TRIzolВ® reagent-based protocol. One Вµg of RNA was handled to obtain cDNA using the High-capacity cDNA Reverse Transcription Kit (Thermo Fisher, Palo Alto, CA).
Barça 85-78 UCAM Murcia: Winning start to Liga Endesa (85 FC Barcelona got off to a winning that in the Liga ACB, but had to work for
it against a tough-spirited UCAM
T315I and VIC-labeled probe for wild-type ABL1. For the dPCR reaction, 6. 25 μL of the second round of the nested PCR were mixed with 0. 75 μL of the 20X TaqMan® assay and 7. 5 μL of 2 × QuantStudio 3D Master Mix, in a 15 μL reaction volume. Then, 14. 5 μL were loaded onto QuantStudio 3D Digital PCR 20 K chips. The cycling conditions were as follows: initial denaturation at 96 °C for 10 min, followed by 39 cycles at 56 °C for 2 min and 98 °C for 30 s and an elongation step of 60 °C for 2 min, always with the cover temperature at 70 °C.
Murcia city adopts popular European e-motorbike rental service To rent a scooter, you must be of legal age and hold an A1 or B licence,
... parking areas which will be outlined in the App or
The patient died 3 months later (Fig. 5C). Patient ALL_30 was diagnosed of Ph-positive ALL, with BCR::ABL1 levels of 38% and no mutations. After 27 months with imatinib and dasatinib with undetectable BCR::ABL1 levels, the patient relapsed and the p. T315I mutation was detected by both techniques (Fig. 5D). Treatment was switched to ponatinib, with levels of BCR::ABL1 below 1.
3). This threshold improved the previous value established by other studies19, 20, even after correcting the VAF by the BCR::ABL1 levels, allowing the detection of mutations even with BCR::ABL1 < 1%. The sensitivity of the test compared to RNA-NestedNGS was 92%, and the specificity was 81. 6%. Most of false positive cases occur in samples with low BCR::ABL1 ratios, confirming the limitations of RNA-based approaches for low tumor burden19 (Fig. 4). The correlation between DNA and RNA implies that resistant mutations mainly occur in the rearranged BCR::ABL1 allele. The applicability of the DNA-DeepNGS method was 100%, being suitable for studying mutations in all samples, whereas up to 5% of the RNA samples did not present the minimum quality requirements for downstream analysis, defined by qPCR of housekeeping genes.
3A, bottom). Triplicates were distinguished between them by adding custom tags at the beginning of the primers. Each of these primers generated three types of reads of the same region after PCR amplification and sequencing that we classified as P1, P2 and P3. This allowed us to compare results between replicates and to eliminate artifacts.
The number of occurrences of the wild-type and the mutation sequences allowed us to compute the VAF ratio (mutated reads/total reads (mutated + non-mutated)). Then, the VAF ratio was compared with the LOD and LOQ calculated for each hotspot independently in 3 triplicated samples of 6 healthy donors. LOD was computed as the mean ratio in control samples plus 3.
Despite our workflow being designed to screen the 1, 129 genetic positions of exons 4–10 covered by the 9 amplicon design, for this work we selected the 36 most prevalent mutations with known clinical outcome31, 32, 33. Biological triplicates to control amplification errors during PCR cycles were analyzed independently by our bioinformatic pipeline (Fig. 3A). To mitigate noise effects, the median of the three replicates was selected as the final ratio value. Design of triplicates was conducted bioinformatically with an in-house workflow designed in R and Bash (Fig.
90-69: Eighth win on the bounce 11/05/2022. Edu Bueno. Photographer: Pedro Castillo. Real Madrid put on a
show against UCAM Murcia in the penultimate Regular Season game.
Figure 5Time course for the mutations measured by DNA-DeepNGS and RNA-NestedNGS, and BCR::ABL1 levels present in the most clinically relevant patients. Y-axis represents the value of RNA-NestedNGS VAF corrected by the ratio BCR::ABL1 (green), DNA-DeepNGS VAF (blue) or ratio BCR::ABL1/ABL1 (red). ALL acute lymphoblastic leukemia, Asci asciminib, B bosutinib, CML chronic myeloid leukemia, D dasatinib, I imatinib, N nilotinib, Niv nivolumab, P ponatinib, R relapse, VAF variant allele frequency.
In total, 25% samples (40/162) presented point mutations in the BCR::ABL1 KD. All samples were analyzed in duplicate, corrected VAF (variant allele frequency) calculated as the value of the VAF multiplied by the levels of BCR::ABL1) showed a Pearson correlation of biological duplicates for the in-house RNA-based method of 0. 806 (p < 0. 001, Fig. 2B). The LOD (limit of detection) of the RNA-NestedNGS is 1. 0E−5 (1. 0E−2 defined by the Thermo Fisher platform plus 1. 0E−3 which is the minimum BCR::ABL1 ratio level to perform the analysis). Regarding CML samples, 31% (27/86) presented KD mutations (6/37 in warning and 21/49 in failure).
S2B). DiscussionWe here report the first approach for BCR::ABL1 KD mutation screening, using DNA as source material. The study demonstrates that the DNA-deepNGS method is feasible, robust and reproducible and can be easily implemented in the laboratory routines. The identification of acquired ABL1 KD point mutations is crucial to anticipate and overcome TKI resistance. Despite SS being the gold standard for KD mutation screening, NGS and dPCR are emerging as more sensitive techniques to detect minor subclones. However, both approaches present certain limitations. The dPCR method is limited to 10–15 mutations per experiment, and mRNA-based NGS presents a high error rate induced by the retro-transcription and amplification steps.
FC Barcelona B 5–0 CD Castellón: Big win in last game at The reserves will not be going into the promotion play-offs and will now
end 2021/22 next Saturday at the ground of UCAM
T315I and other double mutations and could be an exciting option for multi-resistant patients in combination with ponatinib23. Our results also confirmed that most of the mutations below 20% VAF are missed by SS. Although access to NGS for some laboratories could be difficult, it is recommended to study mutations using more sensitive techniques24. On the other hand, the dPCR is more sensitive than the two alternatives shown here25. However, this technique cannot detect de novo mutations and is limited to a handful of mutations.
ResultsScreening of BCR::ABL1 KD mutation by RNA-NestedNGSA total of 162 samples corresponding to 67 CML (86 samples) and 62 ALL patients (76 samples) were studied (Fig. 1). All samples were screened with an in-house RNA-NestedNGS method designed to amplify the entire KD of the rearranged allele and then sequenced by ultra-deep NGS after random enzymatic cleavage (Fig. 2A).
NGS libraries included the sequencing of biological triplicates with an estimated depth of 500, 000x. The generated fastq files were automatically analyzed via a customized bioinformatic pipeline, programmed in Python and R. During the process, firstly, the raw fastq file was pre-processed using the patient information and sample identifiers, run, barcode, amplicon and triplicate identifier. Thus, the file was demultiplexed into smaller fastq files that allowed the computational optimization of the algorithm, significantly reducing the amount of time needed to analyze each sample. Secondly, to compute the variant read frequency ratios of each position in the coding regions of the selected exons, the aligned wild-type and mutated sequences (with a margin of 15 bp, queried to Ensembl34 via its Python application programming interface, API) of each position were searched in the corresponding demultiplexed output file from the previous step.
A two-layer mono-objective algorithm based on guided The main idea of VS is to process an existing database of approved De
Murcia (UCAM), Campus de los Jerónimos, 30107, Murcia,
Former Michigan State player signs in Spain after MVP season in Poland Trice has signed with UCAM Murcia of Spain's Liga Endesa for the 2022-23
season, the club recently