The Impact of DNA Input Amount and DNA Source on the Performance of Whole-Exome Sequencing in Cancer Epidemiology

Zhu, Qianqian; Hu, Qiang; Shepherd, Lori; Wang, Jianmin; Morrison, Carl; Conroy, Jeffrey M.; Glenn, Sean T.; Davis, Warren; Kwan, Marilyn L.; Ergas, Isaac J.; Roh, Janise M.; Kushi, Lawrence H.; Ambrosone, Christine B.; Liu, Song; Yao, Song

doi:10.1158/1055-9965.epi-15-0205

Cited by 29 publications

(29 citation statements)

References 38 publications

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“…For 90.39% of gold SNPs, WES and WGS 420 yielded the same genotype. More than 63.41% of these concordant SNVs were 421 identified as heterozygous, which was similar to those obtained in previous WES 422 studies [4,15,25]. 423…”

Section: Wgs and Wes 391supporting

confidence: 81%

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Characterizing sensitivity and coverage of clinical WGS as a diagnostic test for genetic disorders

Sun

Liu

Fan

et al. 2020

Preprint

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23Background: With the reduce of cost and incomparable advantages, WGS is likely to 24 change the way of clinical diagnosis of rare and undiagnosed diseases. However, the 25 sensitivity and breadth of coverage of clinical WGS as a diagnostic test for genetic 26 disorders has not been fully evaluated, especially for CNV detection. 27Methods: All the samples underwent WGS using MGISEQ-2000. The performance of 28 NA12878, YH cell line, and the Chinese trios were measured for sensitivity, PPV, depth 29 and breadth of coverage. We also compared the performance of WES and WGS using 30 NA12878. The sensitivity and PPV were tested using family-based trio design in the 31 Chinese trios. We also developed a systematic WGS pipeline for the analysis of 8 32 clinical cases with known disease-causing variants. 33Results: In general, the sensitivity and PPV for SNV/indel detection increased with 34 mean depth, and reached a plateau at a ~40X mean depth using down-sampling samples 35 of NA12878. With a mean depth of 40X, the sensitivity of homozyous and 36 heterozygous SNPs of NA12878 was >99.25% and >99.50% respectively, and PPVs 37 were 99.97% and 98.96%. Homozygous and heterozygous indels showed lower 38 sensitivity and PPV. The sensitivity and PPV is still not 100% even with a mean depth 39 of ~150X. We also observed a substantial variation in the sensitivity of CNV detection 40 across different tools, especially in CNVs with a size of less than 1kb. In general, the 41 breadth of coverage for disease-associated genes and CNVs increased with mean depth. 42 3 The sensitivity and coverage of WGS (~40X) is better than WES (~120X). Among the 43 Chinese trios with ~40X mean depth, the sensitivity in the offspring was >99.48% 44 and >96.36% for SNP and indel detection, and PPVs were 99.86% and 97.93%. All the 45 12 previously validated variants in the 8 clinical cases were successfully detected by our 46 WGS pipeline. 47 Conclusions:The current standard of a mean depth of 40X may be sufficient for 48 SNV/indel detection and identification of most CNVs. Clinical scientists should know 49 the range of sensitivity and PPV for different classes of variants for a particular WGS 50 pipeline, which would be useful when interpreting and delivering clinical reports. 51

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Section: Wgs and Wes 391supporting

confidence: 81%

“…87 So far, WGS still follows the three steps since the invention of MPS technology: 88 preparation of template, library construction and sequencing, and data analysis. Factors 89 that influence the three steps (such as quality of genomic DNA [15,16], library 90 preparation [17][18][19], sequencing platform [18,20], bioinformatics analytical pipeline 91…”

mentioning

confidence: 99%

Characterizing sensitivity and coverage of clinical WGS as a diagnostic test for genetic disorders

Sun

Liu

Fan

et al. 2020

Preprint

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“…But it is possible that sample 5 was improperly collected with more oral microbiome contamination at the time of collection. In a similar series of comparisons involving whole exome sequencing, Zhu et al (2015) found that sequences from blood had a 3.3% higher proportion with minimum 20x coverage in blood compared to saliva but this was not significantly different. With randomly downsampling each genomic sequence in order to ensure an equal number of reads between paired samples, there remained a non-significant difference in coverage between blood and saliva genomes, and this difference was lower than the difference reported in exome comparisons by Zhu et al (2015).…”

Section: Discussionmentioning

confidence: 94%

Quality of Whole Genome Sequencing from Blood versus Saliva Derived DNA in Cardiac Patients

Yao

Akinrinade

Chaix

et al. 2019

Preprint

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Whole-genome sequencing (WGS) is becoming an increasingly important tool for detecting genomic variation. Blood derived DNA is the current standard for WGS for research or clinical purposes. We compared the level of microbial contamination, sequencing coverage, as well as yield and concordance of single-nucleotide polymorphism (SNP) and copy number variant (CNV) calls in WGS from paired blood and saliva samples from 5 pediatric heart disease patients. We found that although saliva samples contained a higher proportion of sequence reads that map to the human oral microbiome, these reads were readily excluded by mapping the reads to the human reference genome. Sequencing coverage was low only in 1 of 5 saliva samples. Over 95% SNPs (including rare SNPs) but <80% CNVs called in blood genomes were detected in paired saliva genomes. These findings suggest that most good quality saliva samples can serve as an alternative to blood samples for detection of sequence variants from WGS in cardiovascular disease patients.

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“…Equally importantly, however, the DNA input amount could play an important role in determining data quality. Although we previously evaluated and found little difference in data quality with over ten times difference in DNA input amounts (200 to 3,000 ng) [19], it is possible that the data quality is more sensitive to DNA amount under the current SCS settings with significantly less DNA. In this study, we focused on comparing existing protocols as-is and did not evaluate the effects of the amount of input DNA.…”

Section: Discussionmentioning

confidence: 90%

“…Germline variants, including SNPs and small insertions/deletions (Indels), were identified using GATK with default parameters [18]. The quality of variant calls was evaluated using a previously published method by the heterozygous/ homozygous ratio, the percentage of overlap with dbSNP, and the transition-transversion ratio [19]. Putative somatic mutations were identified by running variation detection module of Bambino using the bulk blood data as control [20].…”

Section: Bioinformatics Data Analysismentioning

confidence: 99%

Comparison of SureSelect and Nextera Exome Capture Performance in Single-Cell Sequencing

Huss

Glenn

et al. 2018

Hum Hered

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Background: Advances in single-cell sequencing provide unprecedented opportunities for clinical examination of circulating tumor cells, cancer stem cells, and other rare cells responsible for disease progression and drug resistance. On the genomic level, single-cell whole exome sequencing (scWES) started to gain popularity with its unique potentials in characterizing mutational landscapes at a single-cell level. Currently, there is little known about the performance of different exome capture kits in scWES. Nextera rapid capture (NXT; Illumina, Inc.) has been the only exome capture kit recommended for scWES by Fluidigm C1, a widely accessed system in single-cell preparation. Results: In this study, we compared the performance of NXT following Fluidigm’s protocol with Agilent SureSelectXT Target Enrichment System (AGL), another exome capture kit widely used for bulk sequencing. We created DNA libraries of 192 single cells isolated from spheres grown from a melanoma specimen using Fluidigm C1. Twelve high-yield cells were selected to perform dual-exome capture and sequencing using AGL and NXT in parallel. After mapping and coverage analysis, AGL outperformed NXT in coverage uniformity, mapping rates of reads, exome capture rates, and low PCR duplicate rates. For germline variant calling, AGL achieved better performance in overlap with known variants in dbSNP and transition-transversion ratios. Using calls from high coverage bulk sequencing from blood DNA as the golden standard, AGL-based scWES demonstrated high positive predictive values, and medium to high sensitivity. Lastly, we evaluated somatic mutation calling by comparing single-cell data with the matched blood sequence as control. On average, 300 mutations were identified in each cell. In 10 of 12 cells, higher numbers of mutations were identified using AGL than NXT, probably caused by coverage depth. When mutations are adequately covered in both AGL and NXT data, the two methods showed very high concordance (93–100% per cell). Conclusions: Our results suggest that AGL can also be used for scWES when there is sufficient DNA, and it yields better data quality than the current Fluidigm’s protocol using NXT.

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The Impact of DNA Input Amount and DNA Source on the Performance of Whole-Exome Sequencing in Cancer Epidemiology

Cited by 29 publications

References 38 publications

Characterizing sensitivity and coverage of clinical WGS as a diagnostic test for genetic disorders

Characterizing sensitivity and coverage of clinical WGS as a diagnostic test for genetic disorders

Quality of Whole Genome Sequencing from Blood versus Saliva Derived DNA in Cardiac Patients

Comparison of SureSelect and Nextera Exome Capture Performance in Single-Cell Sequencing

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