Research output: Contribution to journal › Article › peer-review
An inexpensive, simple and effective method of genome DNA fragmentation for NGS libraries. / Kechin, Andrey; Boldyreva, Darya; Borobova, Viktoriya et al.
In: Journal of biochemistry, Vol. 170, No. 5, 01.11.2021, p. 675-681.Research output: Contribution to journal › Article › peer-review
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TY - JOUR
T1 - An inexpensive, simple and effective method of genome DNA fragmentation for NGS libraries
AU - Kechin, Andrey
AU - Boldyreva, Darya
AU - Borobova, Viktoriya
AU - Boyarskikh, Ulyana
AU - Scherbak, Sergey
AU - Apalko, Svetlana
AU - Makarova, Maria
AU - Mosyakin, Nikolay
AU - Kaftyreva, Lidia
AU - Filipenko, Maxim
N1 - Publisher Copyright: © 2021 The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.
PY - 2021/11/1
Y1 - 2021/11/1
N2 - Next-generation sequencing (NGS)-library preparation for whole-genome sequencing (WGS) starts with DNA fragmentation, and sonication is a physical approach used most often due to its simplicity and reproducibility. However, the commercially available Covaris instrument has a high price for both the device and consumables. Here, we describe our in-house method of DNA shearing by sonication with small (100-600 µm) glass beads and an ultrasonic bath. The fragmentation conditions were optimized for the bacterial WGS with ∼550-bp fragment size (the ultrasonic bath water temperature 5-10°C, glass beads 0.06 g, the fragmentation time 50 s) and for human DNA with ∼250 bp (fragmentation with the same parameters for 4 min). Fragmentation results were compared with the Covaris instrument for preparing several bacterial NGS libraries for Illumina NGS platforms by several characteristics. We obtained close mean fragment lengths (523-623 versus 480-646), similar mono- and dinucleotide specificity of shearing, and comparable indicators of read alignment and de novo assembly for both methods. Thus, the described method is a new fast, and effective DNA fragmentation approach that can be used in different WGS applications.
AB - Next-generation sequencing (NGS)-library preparation for whole-genome sequencing (WGS) starts with DNA fragmentation, and sonication is a physical approach used most often due to its simplicity and reproducibility. However, the commercially available Covaris instrument has a high price for both the device and consumables. Here, we describe our in-house method of DNA shearing by sonication with small (100-600 µm) glass beads and an ultrasonic bath. The fragmentation conditions were optimized for the bacterial WGS with ∼550-bp fragment size (the ultrasonic bath water temperature 5-10°C, glass beads 0.06 g, the fragmentation time 50 s) and for human DNA with ∼250 bp (fragmentation with the same parameters for 4 min). Fragmentation results were compared with the Covaris instrument for preparing several bacterial NGS libraries for Illumina NGS platforms by several characteristics. We obtained close mean fragment lengths (523-623 versus 480-646), similar mono- and dinucleotide specificity of shearing, and comparable indicators of read alignment and de novo assembly for both methods. Thus, the described method is a new fast, and effective DNA fragmentation approach that can be used in different WGS applications.
KW - DNA fragmentation
KW - glass beads
KW - shearing
KW - sonication
KW - whole-genome sequencing
UR - http://www.scopus.com/inward/record.url?scp=85123226424&partnerID=8YFLogxK
UR - https://www.elibrary.ru/item.asp?id=47558877
U2 - 10.1093/jb/mvab089
DO - 10.1093/jb/mvab089
M3 - Article
C2 - 34382083
AN - SCOPUS:85123226424
VL - 170
SP - 675
EP - 681
JO - Journal of biochemistry
JF - Journal of biochemistry
SN - 0021-924X
IS - 5
ER -
ID: 35322134