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Radiotherapy for localized disease, splenectomy, or a period of observation prior to systemic therapy were permitted.

PFS and overall survival OS were calculated from the date of initiation of systemic therapy. R-CHOP were included in Cox regression multivariate models using a backward likelihood ratio approach.

A total of BR-treated patients were identified. Table 1 shows clinical and treatment characteristics. With a median follow-up of 2. In multivariate analysis including these variables as well as chemoimmunotherapy regimen, treatment with BR was not associated with PFS or OS.

In this population-based analysis, BR is an effective induction regimen for both transplant eligible and ineligible patients. Longer follow-up is necessary to fully understand the impact of BR in the frontline setting.

Sign In or Create an Account. Sign In. Article Navigation. This Site. Google Scholar. A qualified primer was identical to the reference sequence, which was able to amplify the target region blue lines without producing noise sequences from other regions green lines.

In order to count reads accurately, a filtering process was designed to eliminate noise sequences. For noise case 1, the PCR did not amplify any noise product.

For cases 2 and 3, the unique variation patterns blue dots between them and the reference sequence were exploited to filter noise sequences in the bioinformatic analysis step.

When counting single allelic molecules, one significant bias is caused by the noise from the homologous sequences with the target sequences.

During evolution, similar sequences, existing as pseudogenes or homologous genes, reside all over the genome due to chromosome repetition, retrotransposition of mature RNAs, and other genetic events.

First, for the primer design, we intentionally avoided regions with SNPs, as they could have decreased the PCR efficiency and thus skewed the mutation ratios.

Lastly, a bioinformatic filtering algorithm was used to automatically remove all noise sequences. These reference standards served as a valuable testing subject for three reasons: 1 They were commercially available with different mutation ratios.

The maternal genotype could have been either AA homozygous or AB heterozygous. To develop a molecule counting assay and test different conditions for optimization, adequate reference standard material was a necessity.

A Evaluation of gDNA as a reference sample. D Evaluating the correlation between starting DNA and tested unique reads Pearson correlation coefficient analysis.

H A total of 67 samples with HBB :c. As there were no sufficient ABbb samples for statistics, individual dots denoted the detected ratios.

An optimization process containing the primer design and the bioinformatic filtering was exploited to eliminate noise sequences.

Excluding the noise from total molecule counts was critically important because the noise would have significantly skewed the measurements of both mean values and standard deviations.

The elimination of the noise improved the measurement to The improvement of the detected mutation ratios from Losing fragments is inevitable during ligation, cleaning, and transfer.

Therefore, the final unique reads only represented a small portion of the total number of fragments in the starting DNA. We hypothesized that with the same sample, the tested unique reads would be correlated with the starting DNA.

As the tested unique reads were used for calculating the mutation ratios and then deducing genotypes, a certain amount of starting DNA and tested unique reads were required.

Practically, 10 mL maternal blood could contain at least 20 ng cfDNA, which is enough for one test and one backup storage. Therefore, the fetal portion should be large enough for an accuracy measurement.

There were pregnancy samples that were independently tested by cfBEST and IMD, while the results from the latter are adopted as golden standards.

To further assess the sensitivity and specificity of cfBEST, the alleles were also introduced in the concordant analysis.

The concordance rate, which is defined as the ratio of concordant cases among detected genotypes, was For individual alleles, concordance rates are shown in Table 2.

Among all alleles, one false negative 0. Thirteen out of 16 mutations were listed, as three mutations, HBB :c. Currently, prenatal diagnosis mainly relies on invasive procedures that pose associated risks to both the fetus and the pregnant mother.

Ligating cfDNA fragments with UMIs could effectively distinguish PCR duplicates from those original molecules that happened to be fragmented into exactly same starts and ends.

According to the sequencing data produced in our study, — unique fragments were recovered per sample while only — of them are distinguishable by their ends.

Therefore, thanks to UMIs, the valid original molecules capacity per sample could theoretically be increased by tenfold for the same amount of sequencing reads without mistakenly filtering them as PCR duplicates.

Actually, without UMIs the number of recovered unique reads would be much fewer regardless of the sequencing depths. In addition, various reaction conditions such as ionic strength, annealing temperature, primer design, multiplex combination, PCR additives, and PCR rounds were explored to improve the amplification efficiency and the uniformity of multiplexed PCR, leading to a higher amount of recovered target molecules.

The average recovered unique fragments were doubled without increasing sequencing data by introducing the UMI system.

The sequences containing the chosen SNPs encompassed no pseudogenes or homologous genes that could have produced noise sequences capable of skewing the allelic frequencies.

Different from noninvasive prenatal testing for chromosome aneuploidy, which would have a certain number of false positives due to confined placental mosaicism, 31 our precise counting system that directly quantifies cfDNA allelic molecules could become a diagnostic tool instead of a screening method.

All we could rely on is the absolute molecule numbers that reflects the fetal mutation ratios without the prior information of parental genotypes.

When maternal genotypes were homozygous AA , the fetus could either have been aa or ab. To overcome these challenges, we needed to specifically improve the measurement accuracy in detection of heterozygous mutations.

In our experiment, we found several ways to improve the measurement accuracy: 1 excluding noise sequences through primer designing and bioinformatics filtering; 2 increasing the starting amount of DNA; 3 increasing the recovered unique reads; and 4 increasing the cutoff of the fetal DNA fraction.

With these predetermined parameters, we blindly tested genomes and successfully genotyped of them concordance rate The detection of total alleles specific for each of 13 mutation sites from samples achieved a sensitivity of Since we demonstrated cfBEST to be a reliable and accurate method for diagnosing monogenic disorders, we propose guidelines for developing assays based on the cfBEST system.

In our study, this panel was applied to Han Chinese population, so any further development targeting this population could use this panel directly.

Second, specific primers for the mutation sites should be designed to combine with bioinformatic filtering to target the real molecules.

Third, the peripheral blood of mutation carries should be collected to extract the gDNA to optimize the corresponding assay.

Fifth, a sample cohort should be tested, and sensitivity and specificity should be assessed on allele level.

Moreover, cfBEST possesses some specific advantages. First, cfBEST has great expansion capabilities.

Multiplex PCR assay can reportedly have a room with amplicons in a single test, 32 and more than amplicons had been achieved in our previous pilot experiment.

Low number of SNPs introduced in our method could reduce the cost and allow an accurate genotyping of more loci, which makes it a practical approach for developing simultaneous detection of multiple genetic diseases within one panel.

The molecule counting techniques directly detect the allelic ratio and then deduce the genotypes. We managed to correctly genotype The detection for alleles achieved a sensitivity of Since cfBEST has been demonstrated to be a reliable and accurate method for monogenic disorders, we proposed the guidelines for developing assays for other monogenic disorders.

Samples in the optimization study included both genomic DNA samples from white blood cells and cfDNA samples from plasma. The homozygous mutations included four samples: HBB :c.

A cohort of 26 pregnant women with a singleton male fetus were recruited to draw peripheral blood for the purpose of measuring fetal DNA fraction Table S3C, Supporting Information.

Another cohort of 67 pregnant women with a heterozygous mutation of HBB :c. All blood donors had no other conditions except carrying an HBB mutation.

The sample characteristics are summarized in Table S7 in the Supporting Information. The library was then split into two equal portions and separately subjected to two rounds of nested PCR for ten cycles.

The strategy was designed to retrieve all possible allelic fragment templates with different sizes to minimize bias, including some fragments in which the ends were close to the mutation site.

The final allelic ratio was determined from a minimum of uniquely barcoded alleles. The primers met these criteria: 1 All primers were designed out of the common SNP sites.

The authors thank all test subjects in this study and the authors thank Hui Yu for her support in the creation of the illustration.

Funding support was given to X. Here, X. The patents pertaining to the results presented in the paper.

Yang X. Zhuo Song, Email: moc. Xiangmin Xu, Email: nc. National Center for Biotechnology Information , U.

SongГјl Г¶den

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