Carrier testing for severe childhood recessive diseases by next-generation sequencing

May combine two claims.

Concomitant analysis of normalized coverage in batches of samples appears to circumvent the need for adjustment for GC content, allowing more accurate detection of segmental losses.

“However, concomitant analysis of normalized coverage in batches of samples appears to circumvent the need for adjustment for GC content (32), allowing more accurate detection of segmental losses.”

• cites [32] sourceDisputed

  The Results state that each sample received its own GC-dependent weighting before its coverage was divided by an average across samples. The Methods likewise describe normalizing a GC-corrected distribution by a multi-sample baseline. Thus, the paper does not present batch normalization as circumventing GC adjustment; it uses both steps. (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

In genome research, an average sequence coverage depth of 30-fold has been accepted as sufficient for detecting single-nucleotide polymorphisms and nucleotide insertions or deletions.

“In genome research, an average depth of sequence coverage of 30-fold has been accepted as sufficient for single-nucleotide polymorphism (SNP) and nucleotide insertion or deletion (indel) detection (15–22).”

• cites [15] sourcePartly supported

  The Results (Accuracy of SNP detection; page not provided) say SNP-detection sensitivity is mainly limited by sequence coverage. The paper does not identify 30-fold average coverage as a sufficient threshold, and the Methods (Computational methods; page not provided) state that insertions and deletions were not considered. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [16] sourcePartly supported

  The Results and Discussion section on coverage uniformity reports that 25-fold average coverage gave at least five reads for about 90% of targeted bases, and the paper evaluates SNP calls. It does not state that 30-fold coverage is an accepted sufficiency threshold or establish that depth for indel detection. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [17] sourcePartly supported

  The paper reports SNP and indel detection in exomes with average coverage of 51× (Abstract; page unavailable). Its Methods specify at least 8× coverage for variant calling, but our copy does not say that an average depth of 30× is an accepted sufficient threshold for detecting both SNPs and indels. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [18] sourcePartly supported

  The Conclusion (page unavailable) says average coverage of at least 20× appears the safest strategy, supporting the general point that substantial coverage aids variant detection. The paper does not identify 30× as an accepted threshold or establish its sufficiency for both SNP and indel detection; its quantified detection results concern SNPs. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [19] sourceCould not verify

  The Methods (Read mapping and variant analysis; page unavailable) use a ≥8× coverage cutoff for sequence calls and describe coding-indel identification, but do not assess whether 30-fold average coverage is sufficient. The Discussion (page unavailable) notes that low coverage can cause missed variant calls; searches for 30-fold coverage and its sufficiency found no statement of the claimed standard. (LLM, conf=0.76)

  Experimental — accuracy not yet measured
• cites [20] sourcePartly supported

  Table 1 reports 27.83-fold coverage for the Korean genome, and the Methods Summary says SNPs and indels were identified. That supports detection at approximately 30-fold coverage in this study, but the paper does not establish that 30-fold is an accepted general threshold for sufficient detection; the main text also reports indel-detection sensitivity below 80%. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [21] sourceCould not verify

  The Online Methods section on SNP detection describes read-count and quality filters, while the RRBS alignment section describes indel mapping; neither says that an average coverage depth of 30-fold is sufficient for detecting both. Searches for 30-fold coverage and a sufficiency threshold found no passage establishing that claim in our copy. (LLM, conf=0.74)

  Experimental — accuracy not yet measured
• cites [22] sourcePartly supported

  The passage describing the family's sequencing (section labelled Abstract; page unavailable) reports an average coverage of 40× and identification of single-nucleotide variants. It does not establish that 30× coverage is accepted as sufficient for detecting SNPs and indels, or report indel detection. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim. May combine two claims.

An American College of Medical Genetics report on expanded newborn screening gives criteria that include test accuracy and cost, disease severity, highly penetrant recessive inheritance, and availability of an intervention.

“Criteria included test accuracy and cost, disease severity, highly penetrant recessive inheritance, and whether an intervention was available for those identified.”

• cites [14] sourcePartly supported

  In “The screening test: availability and characteristics” (page unavailable), the report includes test sensitivity, specificity, and cost among its criteria. “Clinical characteristics of the condition” addresses disease burden, and “Diagnosis, follow-up, treatment, and management” addresses treatment availability. The cited criteria do not establish highly penetrant recessive inheritance as a requirement. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

May combine two claims.

For array-based screening to be well suited, most carriers must be accounted for by a few mutations, and most disease mutations must be nucleotide substitutions.

“To be well suited for array-based screening, however, most carriers must be accounted for by a few mutations, and most disease mutations must be nucleotide substitutions (8, 27, 28).”

• cites [8]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [27] sourceCould not verify

  The Correspondence and Advanced search tools sections describe mutation types in HGMD, but neither discusses when array-based carrier screening is well suited. Searches for array screening and carrier frequency found no passage saying that a few mutations must account for most carriers or that most disease mutations must be nucleotide substitutions. (LLM, conf=0.72)

  Experimental — accuracy not yet measured
• cites [28]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

Target capture and next-generation sequencing have shown efficacy and, recently, scalability for resequencing human genomes and exomes.

“Target capture and next-generation sequencing (NGS) have shown efficacy and, recently, scalability for resequencing human genomes and exomes, providing an alternative potential paradigm for comprehensive carrier testing (15–22).”

• cites [15] sourcePartly supported

  The Results (Capturing and sequencing exon targets; Reproducibility, pages unavailable) demonstrate capture and next-generation sequencing of more than 15,000 human coding exons, with high-confidence genotypes across most of the targeted space. The introductory text reports that whole human genomes had been sequenced with next-generation instruments. The paper does not demonstrate target capture at whole-exome or whole-genome scale, so the scalability claim is supported for large targeted subsets rather than those complete targets. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [16] sourcePartly supported

  The Abstract and scale-up Results report effective next-generation sequencing after targeted enrichment of human genomic regions, scaled to 1.49 Mb. The Discussion describes larger-scale exon capture reported by other studies. This paper does not demonstrate scalable capture and resequencing of whole human genomes or exomes; its own experiments cover subsets of them. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [17] sourcePartly supported

  The paper’s Abstract reports targeted capture and massively parallel sequencing of 12 human exomes, with sensitive variant detection across more than 300 Mb of coding sequence. Its discussion describes the approach as reproducible and able to extend a given amount of sequencing across more samples, supporting efficacy and scalability for exomes. It does not demonstrate resequencing whole human genomes. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [18] sourcePartly supported

  The Abstract and Conclusion report effective target capture and NGS for sequencing exomes from eight people. They do not demonstrate scalability to large cohorts or whole-genome resequencing; the Introduction says full-genome sequencing of many people remains prohibitively costly. Page numbers are unavailable in our copy. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [19] sourceSupported
  Experimental — accuracy not yet measured
• cites [20] sourcePartly supported

  The main text reports massively parallel sequencing of one human genome and high-depth sequencing of selected regions using BAC clones; the Methods Summary gives a six-week run time using three instruments. This supports the feasibility of next-generation whole-genome sequencing, but the paper does not demonstrate exome resequencing, target capture, or scalability across multiple genomes. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [21] sourcePartly supported

  The Main section reports high-coverage, near-complete whole-genome sequencing of two human twins, supporting the efficacy of next-generation sequencing for human genomes. The paper does not establish target capture or exome resequencing, or demonstrate scalability beyond the genomes studied (searched: target capture, exome, scalability). (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [22] sourcePartly supported

  The paper’s sequencing-method and family-analysis passages (section labeled Abstract; page unavailable) describe short-read sequencing of four complete human genomes and its effectiveness in narrowing candidate genes for two disorders. They do not demonstrate target capture, exome resequencing, or scalability beyond this family quartet. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

May combine two claims.

Cost and the absence of accurate, sensitive, scalable technologies have been important barriers to extending preconception screening to most severe recessive mutations or to the general population.

“Hitherto, important criteria precluding extension of preconception screening to most severe recessive mutations or the general population have been cost [defined in that report as an overall analytical cost requirement of <$1 per test per condition (14)] and the absence of accurate, sensitive, scalable technologies.”

• cites [14] sourcePartly supported

  In “The screening test: availability and characteristics” (page unavailable), the report values low-cost, high-throughput testing and sets a criterion of less than $1 per infant screened. It does not state an overall analytical cost requirement of less than $1 per test per condition; its criteria concern newborn screening, not preconception screening. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

May combine two claims.

Variant calling required more than 10 uniquely aligned reads with quality score above 20 and more than 14% of reads supporting the variant.

“Given the need for highly accurate carrier detection, we required >10 uniquely aligned reads of quality score >20 and >14% of reads to call a variant (20, 21).”

• cites [20] sourcePartly supported

  The Methods Summary (page not available) says sequences and runs were used if their average Q scores were at least 20, and that SNPs and indels were identified using optimized filters. It does not specify more than 10 uniquely aligned reads or more than 14% variant-supporting reads; its average-quality criterion is also not the claimed per-read threshold. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [21] sourcePartly supported

  The Methods Summary (page unavailable) reports 11–44× coverage and Q ≥ 20 for genotyping, while the Online Methods describe filters based on uniquely aligning reads and the percentage of reads calling a SNP. Our copy does not establish the claimed combined rule of >10 uniquely aligned reads, quality score >20, and >14% variant-supporting reads; the Methods Summary instead mentions ≥1 uniquely aligning read and refers to supplementary frequency cutoffs. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Substitutions, indels, splicing mutations, and gross deletions account for the vast majority, 96%, of annotated mutations.

“Notably, substitutions, indels, splicing mutations, and gross deletions account for the vast majority (96%) of annotated mutations (27).”

• cites [27] sourceSupported

May combine two claims.

Preliminary experiments suggested that Agilent SureSelect hybrid capture and RainDance microdroplet polymerase chain reaction, but not Febit HybSelect microarray-based capture or Olink padlock probe ligation and PCR, yielded consistent target enrichment.

“Preliminary experiments suggested that existing protocols for Agilent SureSelect hybrid capture (15) and RainDance microdroplet polymerase chain reaction (PCR) (16) but not Febit HybSelect microarray-based biochip capture (30) or Olink padlock probe ligation and PCR (31) yielded consistent target enrichment.”

• cites [15] sourceSupported
• cites [16] sourceSupported
• cites [30] sourcePartly supported

  The Microarray design and synthesis section (p. 2) identifies a febit Geniom microfluidic array, and Figure 1 (p. 3) describes probes capturing PCR products for detection. The paper does not identify this method as HybSelect or assess whether it yielded consistent target enrichment in the citing authors’ preliminary experiments. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

May combine two claims.

The Human Gene Mutation Database lists 102,433 disease mutations, and this number is steadily increasing.

“Given that the Human Gene Mutation Database (HGMD) lists 102,433 disease mutations (27), a number that is steadily increasing, a fixed-content method appeared impractical.”

• cites [27] sourcePartly supported

  The Correspondence section says HGMD records disease-causing mutations and that its annual number of new entries has been steadily increasing. Our copy does not report the claimed total of 102,433 disease mutations; the exact figure is therefore unsupported by this citation. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Previous studies identified copy number variations based on changes in regional coverage along a chromosome in an individual sample.

“Previous studies have identified CNVs on the basis of changes in regional coverage along a chromosome in an individual sample (20, 21).”

• cites [20] sourceSupported
• cites [21] sourcePartly supported

  The Online Methods section on array-based genotyping and CNV detection (page unavailable) confirms that the study identified CNVs in individual DNA samples. It says detection used changes in array-probe intensity and SNP allele ratios, not regional sequencing coverage along a chromosome; that specific method is not supported. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

May combine two claims.

At high volume, the overall analytical cost of the hybrid enrichment-based test was $378, achieving <$1 per test per condition and approximating treatment cost per U.S. live birth.

“At high volume, the overall analytical cost of the hybrid enrichment-based test was $378, achieving the requirement of <$1 per test per condition and approximating that expended on treatment of severe recessive childhood disorders per U.S. live birth (14, 29).”

• cites [14] sourcePartly supported

  The screening-test criteria section specifies a cost criterion of less than $1 per infant screened, and the changing technological landscape section says multiplex testing can reduce cost per condition. Neither section establishes a less-than-$1-per-condition requirement or shows that a $378 hybrid enrichment-based test meets it. The copy also does not give the $378 figure or the claimed comparison with treatment expenditure per U.S. live birth. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [29] sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Microdroplet PCR should theoretically achieve a sensitivity of about 99%, albeit at higher cost.

“Alternatively, microdroplet PCR should theoretically achieve a sensitivity of ~99%, albeit at higher cost (16, 47).”

• cites [16] sourcePartly supported

  The Discussion reports that more than 99.6% of amplicons were successfully amplified, which supports a roughly 99% figure for amplification success, not necessarily sensitivity. It separately reports a 94.5% variant-detection rate for microdroplet PCR. Our copy does not establish the claimed higher cost relative to an alternative. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [47] sourcePartly supported

  The Discussion (page unavailable) reports correct variant calls at up to 99.5% of uniquely mappable positions in the targeted regions, supporting a near-99% figure for this method. It does not describe that figure as a theoretical sensitivity or state that microdroplet PCR costs more; searches for sensitivity and cost found no such comparison. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The 104 DNA samples included 26 well-characterized normal individuals and two previously genome-sequenced individuals.

“Twenty-six samples were well characterized, from “normal” individuals, and two had previously undergone genome sequencing (21).”

• cites [21] sourceCould not verify

  The Main section reports whole-genome sequencing of two twins in this study, but does not say they were previously genome-sequenced members of a 104-sample set. Searches for the 104 samples and 26 well-characterized normal individuals found no passage addressing those details in our copy. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

The distribution of mutations among the sequenced samples appeared random.

“The distribution of mutations among sequenced samples appeared random.”

May combine two claims.

In the reported carrier screen, average target coverage was 160-fold and 93% of nucleotides had at least 20-fold coverage.

“At a resultant 160× average target coverage, 93% of nucleotides had at least 20× coverage”

May combine two claims.

Read pair assembly before alignment with the Illumina HiSeq SBS platform generated longer reads with high-quality scores, with a combined read length of 148.6 ± 3.8 nucleotides and an increase in nucleotides with quality score above 30 from 75% to 83%.

“First, with the Illumina HiSeq SBS platform, we used the novel approach of read pair assembly before alignment (99% efficiency) to generate longer reads with high-quality scores (148.6 ± 3.8 nt combined read length and increase in nucleotides with quality score >30 from 75 to 83%).”

May combine two claims.

As sequence depth increased from 0.7 to 2.7 gigabases, sensitivity increased from 93.9% to 95.6%, while positive predictive value remained about 100%.

“As sequence depth increased from 0.7 to 2.7 Gb, sensitivity increased from 93.9 to 95.6%, whereas PPV remained ~100% (Fig. 6A).”

May combine two claims.

After hybrid capture, the aligned sequence coverage distribution was unimodal but flat and right-skewed.

“The aligned sequence coverage distribution was unimodal but flat (platykurtic) and right-skewed (Fig. 2B).”

May combine two claims.

The requirement for at least 14% of reads to call a variant was highly effective for nucleotides with very high coverage and was derived from the genotype data.

“The requirement for ≥14% of reads to call a variant was highly effective for nucleotides with very high coverage and was derived from the genotype data discussed below.”

May combine two claims.

The positive predictive value of sequence-based SNP genotypes was 99.96% and the negative predictive value was 98.5%, as ascertained by array hybridization.

“The positive predictive value (PPV) of sequence-based SNP genotypes was 99.96% and negative predictive value (NPV) was 98.5%, as ascertained by array hybridization.”

May combine two claims.

Targets with low coverage below 3-fold were highly reproducible and had high GC content.

“Targets with low (<3×) coverage were highly reproducible and had high GC content (table S6).”

May combine two claims.

Primer synthesis failures caused linear amplification of a subset of targets, about 5% of target nucleotides with zero coverage, and a similar proportion of nucleotides on target to the best hybrid capture experiments.

“This resulted in linear amplification of a subset of targets, ~5% of target nucleotides with zero coverage and a similar proportion of nucleotides on target to that obtained in the best hybrid capture experiments (~30%; Table 1).”

May combine two claims.

For known substitution, indel, splicing, gross deletion, and regulatory alleles in 76 samples, sensitivity was 100%, with 113 of 113 known alleles detected.

“For known substitution, indel, splicing, gross deletion, and regulatory alleles in 76 samples, sensitivity was 100% (113 of 113 known alleles; table S7).”

May combine two claims.

Bar code nucleotides did not appreciably interfere with hybrid selection; the stoichiometry of multiplexed pools was essentially unchanged before and after hybrid selection.

“Interference of bar code nucleotides with hybrid selection did not occur appreciably: The stoichiometry of multiplexed pools was essentially unchanged before and after hybrid selection.”

May combine two claims.

Microdroplet PCR gave a coverage distribution narrower than hybrid capture but with similar right skewing.

“In practice, the coverage distribution was narrower than hybrid capture but with similar right skewing (Fig. 2D).”

May combine two claims.

For heterozygote detection, the more-than-10-read requirement was equivalent to about 20-fold coverage, which was achieved in about 96% of exons with about 2.6 gigabases of sequence.

“For heterozygote detection, for example, this was equivalent to ~20× coverage, which was achieved in ~96% of exons with ~2.6 gigabases (Gb) of sequence (Fig. 2C).”

May combine two claims. The quote only partly states this claim.

Hybrid capture achieved at least 30% of nucleotides on target after one round of bait redesign for underrepresented exons and decreased bait representation in over-represented exons.

“This was achieved with hybrid capture after one round of bait redesign for underrepresented exons and decreased bait representation in over-represented exons (Table 1).”

May combine two claims.

With these cutoffs and a requirement for 20-fold coverage and 10 reads of quality at least 20 to call a variant, sequence-based SNP genotyping had 98.8% accuracy, 94.9% sensitivity, and 99.99% specificity.

“With these cutoffs and a requirement for 20× coverage and 10 reads of quality ≥20 to call a variant, the accuracy of sequence-based SNP genotyping was 98.8%, sensitivity was 94.9%, and specificity was 99.99%.”

May combine two claims.

In sample NA02057, next-generation sequencing found homozygosity for two adjacent AGA substitutions, of which C163S had been shown to be the disease mutation.

“NA02057, from a female with aspartylglucosaminuria (OMIM #208400), characterized as a compound heterozygote, was homozygous for two adjacent substitutions (AGA exon 4, c.482G>A, R161Q, chr4:178596918G>A and exon 4, c.488G>C, C163S, chr4:178596912G>C in 38 of 39 reads; Fig. 8), of which C163S had been shown to be the disease mutation (43).”

• cites [43] sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Median coverage increased linearly with sequence depth.

“As expected, median coverage increased linearly with sequence depth.”

May combine two claims.

The proportion of bases with greater than zero coverage and with greater than 20-fold coverage increased toward asymptotes of about 99% and about 96%, respectively.

“The proportion of bases with greater than zero and >20× coverage increased toward asymptotes at ~99 and ~96%, respectively (Table 1 and Fig. 2C).”

May combine two claims.

No significant differences in total carrier burden were found between Caucasians and other ethnicities, between males and females, or between affected and unaffected individuals after correction for compound heterozygosity in affected individuals.

“However, whereas group sizes were small, no significant differences in total carrier burden were found between Caucasians and other ethnicities, between males and females, nor between affected and unaffected individuals (after correction for compound heterozygosity in those affected).”

May combine two claims.

The requirement for at least 10 high-quality reads to substantiate a variant call resulted in 99.96% specificity for single-nucleotide substitutions and 100% specificity for about 200 known mutations and new indels.

“The requirement for at least 10 high-quality reads to substantiate a variant call resulted in a specificity of 99.96% for single-nucleotide substitutions (which is the limit of accuracy for the gold standard method used) and 100% for about 200 known mutations and new indels in our screening method.”

May combine two claims.

437 genes representing 507 recessive diseases met inclusion criteria, of which 448 diseases were severe.

“Four hundred and thirty-seven genes, representing 507 recessive diseases, met these criteria, of which 448 diseases were severe (table S3).”

Probably not a substantial claim: may not pass S4 (based). May combine two claims.

Current reference resources contain common polymorphisms annotated as disease mutations and erroneous disease mutations.

“Specifically, current reference resources contain common polymorphisms that are annotated as disease mutations and erroneous disease mutations.”

May combine two claims.

74% of disease mutation calls were accounted for by 47 substitutions each with incidence ≥5%, and 20 of these were homozygous in samples unaffected by the corresponding disease.

“Seventy-four percent of these, however, were accounted for by 47 substitutions each with an incidence of ≥5%, of which 20 were homozygous in samples unaffected by the corresponding disease (table S8).”

• Also stated in: § DISCUSSION · plain

  “Unexpectedly, we found that 74% of “disease mutation” calls were accounted for by substitutions with incidences of ≥5%, of which almost one-half were homozygous in samples unaffected by the corresponding disease.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The burden of nonconservative, nonsynonymous, uncommon (<5% incidence) VUS was about 11 per sample.

“The burden of nonconservative, nonsynonymous, uncommon (<5% incidence) VUS was ~11 per sample.”

May combine two claims.

Specificity was 99.96%, and sensitivity of about 95% was achieved with hybrid capture at a sequence depth of 2.5 Gb per sample.

“Specificity was 99.96%, and a sensitivity of ~95% was attained with hybrid capture at a sequence depth of 2.5 Gb per sample.”

Probably not a substantial claim: may not pass S4 (based).

Comprehensive mutation sets, population allele frequencies, and individual mutation genotype-phenotype relationships have been defined for very few recessive diseases.

“Second, comprehensive mutation sets, allele frequencies in populations, and individual mutation genotype-phenotype relationships have been defined in very few recessive diseases.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Among 7028 disorders suspected to have Mendelian inheritance, 1139 are recessive and have a known molecular basis.

“Of 7028 disorders with suspected Mendelian inheritance, 1139 are recessive and have an established molecular basis.”

• Also stated in: § INTRODUCTION · plain

  “Over the past 25 years, 1139 genes that cause Mendelian recessive diseases have been identified (8).”

• cites [8]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Mendelian diseases together cause about 20% of infant deaths and about 10% of pediatric hospitalizations.

“Although individual Mendelian diseases are uncommon in general populations, collectively, they account for ~20% of infant mortality and ~10% of pediatric hospitalizations (6, 7).”

• cites [6] sourceNot checked — no full text of the source
• cites [7] sourceNot checked — no full text of the source

Classified as probably not a substantial claim. May combine two claims.

A recent workshop recommended high analytical validity, concordance across many settings, high throughput, and cost-effectiveness for qualifying new methodologies for broader population-based carrier screening.

“A recent workshop provided recommendations for qualification of new methodologies for broader population-based carrier screening (23). These were high analytical validity, concordance in many settings, high throughput, and cost-effectiveness (including sample acquisition and preparation).”

• cites [23]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Classified as probably not a substantial claim.

Preconception screening with genetic counseling of carriers has led to large reductions in the incidence of several severe recessive diseases, including Tay-Sachs disease and cystic fibrosis.

“Preconception screening, together with genetic counseling of carriers, has resulted in remarkable declines in the incidence of several severe recessive diseases including Tay-Sachs disease and cystic fibrosis.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing). May combine two claims.

The selected genes are a good representative set but require further assessment of clinical readiness before translation into clinical testing.

“Note that these genes, although a good representative set, require further assessment of clinical readiness before translation into clinical testing.”

Probably not a substantial claim: may not pass S4 (based).

Extending preconception screening to most severe disease genes had been impractical before this work.

“However, extension of preconception screening to most severe disease genes has hitherto been impractical.”

Classified as probably not a substantial claim.

The accuracy of disease mutation genotypes obtained from next-generation sequencing of enriched targets had been uncertain.

“In particular, the accuracy of disease mutation genotypes derived from NGS of enriched targets has been uncertain.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The proportion of targets with at least 20-fold coverage appeared useful for quality assessment.

“The proportion of targets with at least 20× coverage appeared to be useful for quality assessment.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Multiplexing of samples during hybrid selection and next-generation sequencing had not previously been reported.

“Multiplexing of samples during hybrid selection and NGS had not previously been reported.”

Probably not a substantial claim: may not pass S4 (based). May combine two claims.

The number of mismatches tolerated for unique alignment of short-read sequences is highly constrained but increases with read length.

“The number of mismatches tolerated for unique alignment of short-read sequences is highly constrained but increases with read length.”

Classified as probably not a substantial claim.

Comprehensive carrier testing also requires detection of polynucleotide indels, gross insertions, gross deletions, and complex rearrangements.

“However, comprehensive carrier testing also requires detection of polynucleotide indels, gross insertions, gross deletions, and complex rearrangements.”

May combine two claims.

Array-based screening for recessive disorders raised concerns about type 1 errors without confirmatory testing and type 2 errors for disease mutations other than substitutions.

“Other concerns with array-based screening for recessive disorders were type 1 errors in the absence of confirmatory testing and type 2 errors for disease mutations other than substitutions (complex rearrangements, indels, or gross deletions with uncertain boundaries).”

Probably not a substantial claim: may not pass S4 (based).

The vast majority of disease mutations are single-nucleotide substitutions or small indels.

“The vast majority of disease mutations are single-nucleotide substitutions or small indels.”

May combine two claims.

Gross deletions were detected both by perfect alignment to mutant junction reference sequences and by local decreases in normalized coverage.

“Third, gross deletions were detected both by perfect alignment to mutant junction reference sequences and by local decreases in normalized coverage (normalized to total sequence generated; C. H. Hu, personal communication).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Omission of gel purification was critical for scalability of library generation.

“Omission of gel purification was critical for scalability of library generation.”

May combine two claims.

Unambiguous alignment of short-read sequences is typically confounded by repetitive sequences, but this was not relevant for carrier testing because the targets overwhelmingly contained unique sequences.

“Unambiguous alignment of short-read sequences is typically confounded by repetitive sequences, but was not relevant for carrier testing, because targets overwhelmingly contained unique sequences.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The Illumina sequencing-by-synthesis and SOLiD sequencing-by-ligation platforms are widely disseminated and have throughput of at least 50 gigabases per run and read lengths of at least 50 nucleotides.

“Of these, the Illumina sequencing-by-synthesis (SBS) and SOLiD sequencing-by-ligation (SBL) platforms are widely disseminated and have throughput of at least 50 Gb per run and read lengths of at least 50 nt.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Targets failing hybrid capture could be predicted and perhaps rescued by individual PCRs.

“This suggested that targets failing hybrid capture could be predicted and, perhaps, rescued by individual PCRs.”

Probably not a substantial claim: may not pass S3 (load-bearing).

A burden of 2.8 per genome agreed with theoretical estimates of reproductive lethal allele burden.

“Nevertheless, a burden of 2.8 per genome agreed with theoretical estimates of reproductive lethal allele burden (56).”

• cites [56]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing).

Comprehensive newborn screening for treatable or preventable Mendelian diseases would allow early diagnosis and treatment while neonates are asymptomatic.

“For example, comprehensive newborn screening for treatable or preventable Mendelian diseases would allow early diagnosis and institution of treatment while neonates are asymptomatic.”

May combine two claims.

Many individuals in the cohort were affected by one of these diseases; although correction was made for compound heterozygote and homozygote alleles, the burden estimate did not correct for other potential selection biases.

“Third, many individuals in our cohort were affected by one of these diseases. Although a correction was made for compound heterozygote and homozygote alleles, the burden estimate did not correct for other potential selection biases.”

May combine two claims.

Community-based carrier testing has had high uptake, without apparent stigma or discrimination, and with substantial reductions in the frequencies of tested disorders.

“Community-based carrier testing has had high uptake, without apparent stigma or discrimination and with substantial reductions in the frequencies of tested disorders (3, 48, 49, 51–54).”

• cites [3]Not checked — no full text of the source
• cites [48] sourceNot checked — no full text of the source
• cites [49] sourceNot checked — no full text of the source
• cites [51]Not checked — no full text of the source
• cites [52] sourceNot checked — no full text of the source
• cites [53]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [54]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S4 (based). May combine two claims.

Combining the technology with genetic counseling could reduce the incidence of severe recessive pediatric diseases and may help expedite diagnosis in newborns.

“Combining this technology with genetic counseling could reduce the incidence of severe recessive pediatric diseases and may help to expedite diagnosis of these disorders in newborns.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The technology platform described is agnostic with regard to target genes or clinical setting.

“Finally, the technology platform described herein is agnostic with regard to target genes or clinical setting.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

Additional refinement of alignment parameters is needed.

“We noted, however, that identification of larger polynucleotide indels was influenced in some sequences by the particular alignment seed, suggesting that additional refinement of alignment parameters is needed.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

Early treatment can have a profound impact on clinical severity and could provide a framework for centralized assessment of investigational new treatments before organ failure.

“Early treatment can have a profound impact on the clinical severity of conditions and could provide a framework for centralized assessment of investigational new treatments before organ failure.”

May combine two claims.

Key advantages of NGS are clonal derivation, maintenance of phase information, and highly redundant coverage.

“Key advantages of NGS are clonal derivation (facilitating unambiguous detection of heterozygous and indel variants), maintenance of phase information (allowing haplotype derivation for adjacent variants), and highly redundant coverage (resulting in extremely low consensus error rates).”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The analytical cost will decrease as NGS throughput improves, but test interpretation, reporting, genetic counseling, and stewardship of mutation databases will confer considerable additional costs.

“Although the analytical cost will decrease as the throughput of NGS improves, test interpretation, reporting, genetic counseling, and stewardship of mutation databases will confer considerable additional costs.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

In some cases, such as Duarte variant galactosemia, molecular testing would be superior to conventional biochemical testing.

“In some cases, such as Duarte variant galactosemia, molecular testing would be superior to conventional biochemical testing.”

May combine two claims.

The study used DNA from EBV-transformed cell lines, which are known to undergo somatic hypermutation.

“However, the current study used DNA from Epstein-Barr virus (EBV)–transformed cell lines in which somatic hypermutation has been noted (45).”

• cites [45] sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

A variety of medical applications for this technology exist beyond preconception carrier screening.

“A variety of medical applications for this technology exist beyond use in preconception carrier screening.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

An adequate compilation of pathogenic mutations does not currently exist, and strong evidence of pathogenicity was absent for some variants referred to as disease mutations.

“First, as discussed, an adequate compilation of pathogenic mutations does not currently exist, and strong evidence of pathogenicity was absent for some of the variants referred to as disease mutations.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The carrier incidence and mutation spectrum of individual recessive disorders vary widely among populations, as exemplified by cystic fibrosis.

“As exemplified by cystic fibrosis, the carrier incidence and mutation spectrum of individual recessive disorders vary widely among populations (46).”

• cites [46] sourceNot checked — no full text of the source

May combine two claims.

The adequacy of hierarchical clustering was supported by identical-twin samples being nearest neighbors and by two disease mutations in linkage disequilibrium being nearest neighbors.

“Adequacy of hierarchical clustering was attested to by samples from identical twins being nearest neighbors, as were two disease mutations in linkage disequilibrium.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

Organ or symptom menu-based diagnostic testing, with masking of nonselected conditions, is anticipated to assist clinical geneticists and pediatric neurologists.

“Organ or symptom menu-based diagnostic testing, with masking of nonselected conditions, is anticipated to assist clinical geneticists and pediatric neurologists, because current practice often involves costly, sequential testing of numerous candidate genes.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Technical standards and guidelines have been established for laboratory-developed genetic testing for rare disorders in accredited laboratories.

“Although technical standards and guidelines have been established for laboratory-developed genetic testing for rare disorders in accredited laboratories (26), there are several challenges in their adoption for NGS and bioinformatic-based testing of ~500 conditions.”

• cites [26]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.