Optimization of enzymatic fragmentation is crucial to maximize genome coverage: a comparison of library preparation methods for Illumina sequencing

Table 1 Mean DNA insert sizes upon fragmentation and after sequencing achieved with different library preparation kits

Kit	Target insert size [bp]	10 ng input				100 ng input
		frag. time [min]	Insert size [bp]		PCR cycles	frag. time [min]	Insert size [bp]		PCR cycles
		frag. time [min]	by Tapestation	by seq. Reads	PCR cycles	frag. time [min]	by Tapestation	by seq. Reads	PCR cycles
Nextera DNA flex (Illumina)	450	15*	418(±5)	326(±2)	8	15*	479(±2)	366(±2)	5
Kapa HyperPlus (Roche)	350	20	345(±7)	240(±9)	9	20	-	227(±3)	-
SparQ Fragment and Library Prep (Quantabio)	350	16	245(±8)	185(±3)	9	10	-	244(±10)	-
Swift 2S turbo flexible (Swift)	350	10	422(±9)	330(±12)	6	8	-	226(±7)	-
NEBNext Ultra II FS DNA library prep (NEB)	200-450	15	303(±9)	206(±7)	7	15	276(±7)	188(±6)	3

Libraries were prepared from 10 and 100 ng of human NA12878 DNA using enzymatic fragmentation and tagmentation (*) based library prep kits, employing the given fragmentation times and PCR cycles. Bead-based purifications were performed according to the individual instructions of the DNA library preparation kits. Insert DNA sizes were calculated by subtracting the adapter length from the mean fragment size based on Tapestation D1000 profiles and from the sequencing reads upon mapping after trimming of adapters, standard deviation is given in brackets. PCR-free libraries do not migrate properly on Tapestation, due to forked adapters, therefore there is no insert size data based on Tapestation. For additional information, see also Fig. 1 and Additional Fig. 1A

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