Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 8 (1), 1110

Quantification of Massively Parallel Sequencing Libraries - A Comparative Study of Eight Methods

Affiliations

Quantification of Massively Parallel Sequencing Libraries - A Comparative Study of Eight Methods

Christian Hussing et al. Sci Rep.

Abstract

Quantification of massively parallel sequencing libraries is important for acquisition of monoclonal beads or clusters prior to clonal amplification and to avoid large variations in library coverage when multiple samples are included in one sequencing analysis. No gold standard for quantification of libraries exists. We assessed eight methods of quantification of libraries by quantifying 54 amplicon, six capture, and six shotgun fragment libraries. Chemically synthesized double-stranded DNA was also quantified. Light spectrophotometry, i.e. NanoDrop, was found to give the highest concentration estimates followed by Qubit and electrophoresis-based instruments (Bioanalyzer, TapeStation, GX Touch, and Fragment Analyzer), while SYBR Green and TaqMan based qPCR assays gave the lowest estimates. qPCR gave more accurate predictions of sequencing coverage than Qubit and TapeStation did. Costs, time-consumption, workflow simplicity, and ability to quantify multiple samples are discussed. Technical specifications, advantages, and disadvantages of the various methods are pointed out.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Relative concentration estimates obtained using eight quantification methods. All quantifications were performed in duplicates. The mean was normalised by dividing with the mean of the concentration estimate obtained with the TapeStation instrument. Among the 18 Precision ID Identity Panel libraries, six were categorised as “well PCR amplified” (purple boxplots), six as “adapter dimer rich” (red boxplots), and six as “PCR inhibited” (green boxplots). Ion Xpress fragment libraries and NimbleGen capture libraries are shown in yellow and blue plots, respectively. The NimbleGen capture libraries were not quantified by qPCR, since the qPCR assays targeted Ion Torrent adapters. The lower and upper limits of the box correspond to the 0.25 and the 0.75 quartiles, respectively, and the median is indicated as a line within the box. The ends of the whiskers correspond to the most extreme data point within 1.5 times the interquartile range from the ends of the box. Outliers are indicated by dots.
Figure 2
Figure 2
Quantification of synthetic double-stranded oligos. Four dilutions of two synthetic double-stranded oligos consisting of either the Ion Torrent “A” and “P1” adapter sequences (a) or the Illumina “i7” and “i5” adapter sequences (b) were quantified in duplicate with the NanoDrop (formula image), Qubit (formula image), Bioanalyzer (formula image), GX Touch (formula image), TapeStation (formula image), and Fragment Analyzer (formula image). The mean of the measured oligo concentrations were plotted against the concentrations given by the oligo supplier.
Figure 3
Figure 3
Linear regression analysis of quantifications using the TapeStation and the ABI7500 qPCR instrument with the IonLibQuant assay. Three Precision ID Ancestry Panel libraries were diluted 2×, 4×, 8×, 16×, 32×, and 64×. The dilutions were quantified in triplicate. Means of the triplicates were used for the analysis. The line indicates the linear regression model (y = 0.08× + 0.6, R2 = 0.89). The grey area indicates the 95% confidence interval.
Figure 4
Figure 4
Quantification of dilution series of three Precision ID Ancestry Panel libraries using the TapeStation instrument (a) and the ABI7500 qPCR instrument with the IonLibQuant assay (b,c). The c plot is a zoom of the b plot. The libraries were diluted 2×, 4×, 8×, 16×, 32×, and 64×. Boxplot properties are explained in the legend of Fig. 1. Lines represent linear regression lines. Grey areas represent 95% confidence intervals.
Figure 5
Figure 5
Correlations between library concentration estimates and library coverage. A total of 35 Precision ID Ancestry Panel libraries were quantified prior to sequencing using the Qubit (a), TapeStation (b), or ABI7500 qPCR (c) instrument. The ABI7500 was used in combination with the IonLibQuant assay. Linear regression lines (black line) are plotted with 95% confidence interval (grey area). No correlation was observed between concentration estimates and coverage when using Qubit (R2 = 7.4*10−2, p = 0.114) or TapeStation (R2 = 6.7*10−3, p = 0.651), while the correlation obtained with qPCR was R2 = 0.49 and p = 2.53*10−6.

Similar articles

See all similar articles

Cited by 1 PubMed Central articles

References

    1. Borsting C, Morling N. Next generation sequencing and its applications in forensic genetics. Forensic Sci Int Genet. 2015;18:78–89. doi: 10.1016/j.fsigen.2015.02.002. - DOI - PubMed
    1. Buchard A, Kampmann ML, Poulsen L, Borsting C, Morling N. Iso 17025 validation of a next-generation sequencing assay for relationship testing. Electrophoresis. 2016;37:2822–2831. doi: 10.1002/elps.201600269. - DOI - PubMed
    1. Harris G, O’Toole S, George P, Browett P, Print C. Massive parallel sequencing of solid tumours - challenges and opportunities for pathologists. Histopathology. 2017;70:123–133. doi: 10.1111/his.13067. - DOI - PubMed
    1. Kamps, R. et al. Next-generation sequencing in oncology: Genetic diagnosis, risk prediction and cancer classification. Int J Mol Sci18 (2017). - PMC - PubMed
    1. Deurenberg RH, et al. Application of next generation sequencing in clinical microbiology and infection prevention. J Biotechnol. 2017;243:16–24. doi: 10.1016/j.jbiotec.2016.12.022. - DOI - PubMed

MeSH terms

Feedback