Library Construction for Next-generation Sequencing: Overviews and Challenges

Overview

Journal Biotechniques

Specialties Biomedical Engineering
Biotechnology

Date 2014 Feb 8

PMID 24502796

Citations 222

Authors

Steven R Head

H Kiyomi Komori

Sarah A LaMere

Thomas Whisenant

Filip Van Nieuwerburgh

Daniel R Salomon

Phillip Ordoukhanian

Affiliations

Soon will be listed here.

Abstract

High-throughput sequencing, also known as next-generation sequencing (NGS), has revolutionized genomic research. In recent years, NGS technology has steadily improved, with costs dropping and the number and range of sequencing applications increasing exponentially. Here, we examine the critical role of sequencing library quality and consider important challenges when preparing NGS libraries from DNA and RNA sources. Factors such as the quantity and physical characteristics of the RNA or DNA source material as well as the desired application (i.e., genome sequencing, targeted sequencing, RNA-seq, ChIP-seq, RIP-seq, and methylation) are addressed in the context of preparing high quality sequencing libraries. In addition, the current methods for preparing NGS libraries from single cells are also discussed.

Citing Articles

A rapid and efficient zirconia bead-mediated ultrasonic strategy for DNA fragmentation up to 10 kbp.

Fu P, Zhao T, Wei P, Kong T, Qian S, Wang Y RSC Adv. 2025; 15(8):6068-6075.

PMID: 39995453 PMC: 11848711. DOI: 10.1039/d5ra00027k.

Automation-aided construction and characterization of PrsA strains for the secretion of amylases.

Hamburger F, Schlichting N, Eichenlaub M, Costea P, Sauer C, Jenewein S Front Bioeng Biotechnol. 2025; 12:1479626.

PMID: 39917281 PMC: 11798935. DOI: 10.3389/fbioe.2024.1479626.

Integrated analysis of miRNA and mRNA expression profiles in the bursa of Fabricius of specific pathogen-free chickens infected with avian reticuloendotheliosis virus strain SNV.

Zhao Y, Zhang Q, Wang M, Wu B, Zhao S, Wei X Poult Sci. 2025; 104(3):104847.

PMID: 39874788 PMC: 11810829. DOI: 10.1016/j.psj.2025.104847.

Liquid Biopsy and Multidisciplinary Treatment for Esophageal Cancer.

Hoshi Y, Matsuda S, Takeuchi M, Kawakubo H, Kitagawa Y Cancers (Basel). 2025; 17(2).

PMID: 39857978 PMC: 11763614. DOI: 10.3390/cancers17020196.

Comparison of the rhizospheric soil bacteriomes of Oryza sativa and Solanum melongena crop cultivars reveals key genes and pathways involved in biosynthesis of ectoine, lysine, and catechol meta-cleavage.

Mandal M, Ghosh B, Mandal S Funct Integr Genomics. 2025; 25(1):20.

PMID: 39836258 DOI: 10.1007/s10142-025-01536-x.

References

Kurn N, Chen P, Heath J, Kopf-Sill A, Stephens K, Wang S . Novel isothermal, linear nucleic acid amplification systems for highly multiplexed applications. Clin Chem. 2005; 51(10):1973-81. DOI: 10.1373/clinchem.2005.053694. View

Levin J, Yassour M, Adiconis X, Nusbaum C, Thompson D, Friedman N . Comprehensive comparative analysis of strand-specific RNA sequencing methods. Nat Methods. 2010; 7(9):709-15. PMC: 3005310. DOI: 10.1038/nmeth.1491. View

Bhargava V, Ko P, Willems E, Mercola M, Subramaniam S . Quantitative transcriptomics using designed primer-based amplification. Sci Rep. 2013; 3:1740. PMC: 3638165. DOI: 10.1038/srep01740. View

Shiroguchi K, Jia T, Sims P, Xie X . Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes. Proc Natl Acad Sci U S A. 2012; 109(4):1347-52. PMC: 3268301. DOI: 10.1073/pnas.1118018109. View

Hangauer M, Vaughn I, McManus M . Pervasive transcription of the human genome produces thousands of previously unidentified long intergenic noncoding RNAs. PLoS Genet. 2013; 9(6):e1003569. PMC: 3688513. DOI: 10.1371/journal.pgen.1003569. View

Seguin-Orlando A, Schubert M, Clary J, Stagegaard J, Alberdi M, Prado J . Ligation bias in illumina next-generation DNA libraries: implications for sequencing ancient genomes. PLoS One. 2013; 8(10):e78575. PMC: 3812280. DOI: 10.1371/journal.pone.0078575. View

Oyola S, Otto T, Gu Y, Maslen G, Manske M, Campino S . Optimizing Illumina next-generation sequencing library preparation for extremely AT-biased genomes. BMC Genomics. 2012; 13:1. PMC: 3312816. DOI: 10.1186/1471-2164-13-1. View

Zong C, Lu S, Chapman A, Xie X . Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Science. 2012; 338(6114):1622-6. PMC: 3600412. DOI: 10.1126/science.1229164. View

Hafner M, Landgraf P, Ludwig J, Rice A, Ojo T, Lin C . Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing. Methods. 2007; 44(1):3-12. PMC: 2847350. DOI: 10.1016/j.ymeth.2007.09.009. View

10.

Gundry M, Li W, Maqbool S, Vijg J . Direct, genome-wide assessment of DNA mutations in single cells. Nucleic Acids Res. 2011; 40(5):2032-40. PMC: 3300019. DOI: 10.1093/nar/gkr949. View

11.

Ingolia N, Brar G, Rouskin S, McGeachy A, Weissman J . Genome-wide annotation and quantitation of translation by ribosome profiling. Curr Protoc Mol Biol. 2013; Chapter 4:4.18.1-4.18.19. PMC: 3775365. DOI: 10.1002/0471142727.mb0418s103. View

12.

Hashimshony T, Wagner F, Sher N, Yanai I . CEL-Seq: single-cell RNA-Seq by multiplexed linear amplification. Cell Rep. 2012; 2(3):666-73. DOI: 10.1016/j.celrep.2012.08.003. View

13.

Islam S, Kjallquist U, Moliner A, Zajac P, Fan J, Lonnerberg P . Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. Genome Res. 2011; 21(7):1160-7. PMC: 3129258. DOI: 10.1101/gr.110882.110. View

14.

Gifford C, Ziller M, Gu H, Trapnell C, Donaghey J, Tsankov A . Transcriptional and epigenetic dynamics during specification of human embryonic stem cells. Cell. 2013; 153(5):1149-63. PMC: 3709577. DOI: 10.1016/j.cell.2013.04.037. View

15.

Marine R, Polson S, Ravel J, Hatfull G, Russell D, Sullivan M . Evaluation of a transposase protocol for rapid generation of shotgun high-throughput sequencing libraries from nanogram quantities of DNA. Appl Environ Microbiol. 2011; 77(22):8071-9. PMC: 3209006. DOI: 10.1128/AEM.05610-11. View

16.

Hodges E, Xuan Z, Balija V, Kramer M, Molla M, Smith S . Genome-wide in situ exon capture for selective resequencing. Nat Genet. 2007; 39(12):1522-7. DOI: 10.1038/ng.2007.42. View

17.

Raiber E, Beraldi D, Ficz G, Burgess H, Branco M, Murat P . Genome-wide distribution of 5-formylcytosine in embryonic stem cells is associated with transcription and depends on thymine DNA glycosylase. Genome Biol. 2012; 13(8):R69. PMC: 3491369. DOI: 10.1186/gb-2012-13-8-r69. View

18.

Armour C, Castle J, Chen R, Babak T, Loerch P, Jackson S . Digital transcriptome profiling using selective hexamer priming for cDNA synthesis. Nat Methods. 2009; 6(9):647-9. DOI: 10.1038/nmeth.1360. View

19.

Chen Y, Varani G . Protein families and RNA recognition. FEBS J. 2005; 272(9):2088-97. DOI: 10.1111/j.1742-4658.2005.04650.x. View

20.

Licatalosi D, Mele A, Fak J, Ule J, Kayikci M, Chi S . HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature. 2008; 456(7221):464-9. PMC: 2597294. DOI: 10.1038/nature07488. View