Year: 2025
Type: conference-abstract
Abstract: Background Exome sequencing is an indispensable tool in both research and clinical diagnostics, enabling comprehensive analysis of protein-coding regions in the genome. Reliable collection of high-quality DNA from non-invasive sources such as saliva and buccal swabs is critical for optimal sequencing outcomes. The choice of collection tube can affect DNA yield and quality, potentially influencing downstream analysis. This study compares the performance of two commercially available collection systems—OCR-100 (DNA Genotek, CA) and Kolplagene (Kolplast, BR)—in exome sequencing. Methods In this retrospective study, exome sequencing data were evaluated from saliva and buccal swab samples collected using two tube systems: OCR-100 (DNA Genotek, CA) and Kolplagene (Kolplast, BR). Consecutive samples (n=102) obtained during routine exome procedures were included, comprising 49 OCR-100 and 53 Kolplagene samples. Genomic DNA was extracted using the Maxwell 16 system (Promega), and libraries with target enrichment via exome capture were prepared with the XGen Reagents line according to the manufacturer’s instructions (Integrated DNA Technologies). Sequencing was performed on the DNABSEQ G400 platform (MGI Tech, Co.). The study assessed pre analytical variables such as collection-to-processing time, distance from the collection site to the processing facility, and demographic factors including age, sex, and macroregion. Wet bench, sequencing, and quality control metrics were evaluated, and variant calling was performed to identify SNV and INDELs. Variables were presented using medians (range), and statistical analyses—including Wilcoxon tests—were conducted in R, with significance set at p<0.05. Results In the comparative evaluation of the two tube systems – all values expressed as medians (range) – both OCR-100 and Kolplagene showed similar sample demographics and pre analytical factors: Age – 18(4–358) vs. 14(3–416) days (P=0.372); Collection-to-processing time – 7(3–17) vs. 7(3–18) days (P=0.632); and Distance – 6.95e+02(1–2500) vs. 6.25e+02(1–1937) km (P=0.257). Both systems delivered high quality exome sequencing performance. Wet bench metrics revealed a significantly higher gDNA yield in Kolplagene tubes [1.6e+01(2.56–64) ng/µl] than in OCR-100 [9.46(2.14–36) ng/µl] (P=0.002), while library concentration was comparable [68.58(±11.29) vs. 65.53(±10.28) ng/µl, P=0.157]. Sequencing metrics were consistent: Reads passing filters – 1.55e+08(±2.39e+07) vs. 1.51e+08(±2.28e+07) (P=0.395); Target aligned reads – 9.40e+07(±1.36e+07) vs. 9.19e+07(±1.34e+07) (P=0.421); Duplicated reads – 3.25e+07(1.74e+07–5.58e+07) vs. 3.10e+07(1.73e+07–5.89e+07) (P=0.560); and Paired reads – 1.54e+08(±2.39e+07) vs. 1.50e+08(±2.28e+07) (P=0.400). Mapping quality (MQ>40) did not differ [1.41e+08(±2.18e+07) vs. 1.37e+08(±2.08e+07), P=0.414]. Base metrics were similar: Total bases – 2.32e+10(±3.58e+09) vs. 2.26e+10(±3.42e+09) (P=0.395); Bases with Q>30 – 2.13e+10(±3.38e+09) vs. 2.08e+10(±3.22e+09) (P=0.471). Coverage metrics were equivalent (>20× – 98.18(±0.06) vs. 98.18(±0.06)% (P=0.634); >50× – 97.83(97.31–98.01) vs. 97.81(96.92–98.03)% (P=0.663)). Depth and uniformity were comparable: Depth – 259.32(±36.78) vs. 252.88(±36.09)× (P=0.374); and Uniformity – 97.81(±0.09) vs. 97.82(±0.06)% (P=0.596). Finally, variant calling showed no significant differences: Total variants – 2.11e+05(185185–252828) vs. 2.07e+05(184246–259765) (P=0.272); SNPs – 1.67e+05(146911–199158) vs. 1.64e+05(145278–201638) (P=0.220); Heterozygous SNPs – 1.19e+05(102512–144579) vs. 1.18e+05(103631–141873) (P=0.639); Homozygous SNPs – 9.28e+04(78718–108701) vs. 8.72e+04(71719–114812) (P=0.120); and Heterozygous indels – 1.94e+03(±300.20) vs. 1.84e+03(±268.03) (P=0.089). Conclusion Both OCR-100 and Kolplagene tubes provided comparable exome sequencing performance, although the higher gDNA yield with Kolplagene suggests a potential advantage in DNA extraction that may enhance overall sequencing workflows.
Source: Clinical Chemistry
Authors: Gustavo Barra, Letícia Brito, Gabriela Meguerditchian, Anderson Coqueiro, Pedro Mesquita, Nara Pessoa, Rosenelle Benício, Lorena Gama, Ilária Sgardioli, Amanda Lamas, Alessandra Andrade, Graciella Ribeiro, Rafael Jácomo, Lídia Nery
Institution: Sabin Medicina Diagnostica (Brazil)
Language: English
Topic: Genomics and Rare Diseases
Subfield: Genetics
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W4414740384