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Illuminating choices for library prep: a comparison of library preparation methods for whole genome sequencing of Cryptococcus neoformans using Illumina HiSeq.

Rhodes J, Beale MA, Fisher MC - PLoS ONE (2014)

Bottom Line: When comparing the two newer kits, we found little difference in cost and workflow, with the NEBNext Ultra both slightly cheaper and faster than the TruSeq Nano.However, the quality of data generated using the TruSeq Nano DNA kit was superior due to higher coverage at regions of low GC content, and more SNPs identified.Researchers should therefore evaluate their resources and the type of application (and hence data quality) being considered when ultimately deciding on which library prep method to use.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Disease Epidemiology, Imperial College London, London, United Kingdom.

ABSTRACT
The industry of next-generation sequencing is constantly evolving, with novel library preparation methods and new sequencing machines being released by the major sequencing technology companies annually. The Illumina TruSeq v2 library preparation method was the most widely used kit and the market leader; however, it has now been discontinued, and in 2013 was replaced by the TruSeq Nano and TruSeq PCR-free methods, leaving a gap in knowledge regarding which is the most appropriate library preparation method to use. Here, we used isolates from the pathogenic fungi Cryptococcus neoformans var. grubii and sequenced them using the existing TruSeq DNA v2 kit (Illumina), along with two new kits: the TruSeq Nano DNA kit (Illumina) and the NEBNext Ultra DNA kit (New England Biolabs) to provide a comparison. Compared to the original TruSeq DNA v2 kit, both newer kits gave equivalent or better sequencing data, with increased coverage. When comparing the two newer kits, we found little difference in cost and workflow, with the NEBNext Ultra both slightly cheaper and faster than the TruSeq Nano. However, the quality of data generated using the TruSeq Nano DNA kit was superior due to higher coverage at regions of low GC content, and more SNPs identified. Researchers should therefore evaluate their resources and the type of application (and hence data quality) being considered when ultimately deciding on which library prep method to use.

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Related in: MedlinePlus

Uniquely and commonly called SNPs in TruSeq Nano and NEBNext Ultra-prepared isolates.The majority of called SNPs were common to both methods in each isolate. However, a greater number of SNPs were unique to the isolate prepared with the TruSeq Nano method (blue). Venn diagrams were generated using the Venny software [17] of SNPs called using GATK [14], [15].
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pone-0113501-g002: Uniquely and commonly called SNPs in TruSeq Nano and NEBNext Ultra-prepared isolates.The majority of called SNPs were common to both methods in each isolate. However, a greater number of SNPs were unique to the isolate prepared with the TruSeq Nano method (blue). Venn diagrams were generated using the Venny software [17] of SNPs called using GATK [14], [15].

Mentions: The false positive rate for SNP calling is higher in genomes prepared with TruSeq Nano compared to NEBNext Ultra (Table 2); however, this is not the case when calling INDELs (Table 3). Despite this, more filtered, high confidence SNPs were identified in the isolates prepared with the TruSeq Nano DNA kit, compared to those prepared with the NEBNext Ultra kit. Further investigation revealed that more unique SNPs were called in the isolates prepared with the TruSeq Nano kit (Figure 2) suggesting that not all SNPs are accurately called in isolates prepared with NEBNext Ultra.


Illuminating choices for library prep: a comparison of library preparation methods for whole genome sequencing of Cryptococcus neoformans using Illumina HiSeq.

Rhodes J, Beale MA, Fisher MC - PLoS ONE (2014)

Uniquely and commonly called SNPs in TruSeq Nano and NEBNext Ultra-prepared isolates.The majority of called SNPs were common to both methods in each isolate. However, a greater number of SNPs were unique to the isolate prepared with the TruSeq Nano method (blue). Venn diagrams were generated using the Venny software [17] of SNPs called using GATK [14], [15].
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4237435&req=5

pone-0113501-g002: Uniquely and commonly called SNPs in TruSeq Nano and NEBNext Ultra-prepared isolates.The majority of called SNPs were common to both methods in each isolate. However, a greater number of SNPs were unique to the isolate prepared with the TruSeq Nano method (blue). Venn diagrams were generated using the Venny software [17] of SNPs called using GATK [14], [15].
Mentions: The false positive rate for SNP calling is higher in genomes prepared with TruSeq Nano compared to NEBNext Ultra (Table 2); however, this is not the case when calling INDELs (Table 3). Despite this, more filtered, high confidence SNPs were identified in the isolates prepared with the TruSeq Nano DNA kit, compared to those prepared with the NEBNext Ultra kit. Further investigation revealed that more unique SNPs were called in the isolates prepared with the TruSeq Nano kit (Figure 2) suggesting that not all SNPs are accurately called in isolates prepared with NEBNext Ultra.

Bottom Line: When comparing the two newer kits, we found little difference in cost and workflow, with the NEBNext Ultra both slightly cheaper and faster than the TruSeq Nano.However, the quality of data generated using the TruSeq Nano DNA kit was superior due to higher coverage at regions of low GC content, and more SNPs identified.Researchers should therefore evaluate their resources and the type of application (and hence data quality) being considered when ultimately deciding on which library prep method to use.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Disease Epidemiology, Imperial College London, London, United Kingdom.

ABSTRACT
The industry of next-generation sequencing is constantly evolving, with novel library preparation methods and new sequencing machines being released by the major sequencing technology companies annually. The Illumina TruSeq v2 library preparation method was the most widely used kit and the market leader; however, it has now been discontinued, and in 2013 was replaced by the TruSeq Nano and TruSeq PCR-free methods, leaving a gap in knowledge regarding which is the most appropriate library preparation method to use. Here, we used isolates from the pathogenic fungi Cryptococcus neoformans var. grubii and sequenced them using the existing TruSeq DNA v2 kit (Illumina), along with two new kits: the TruSeq Nano DNA kit (Illumina) and the NEBNext Ultra DNA kit (New England Biolabs) to provide a comparison. Compared to the original TruSeq DNA v2 kit, both newer kits gave equivalent or better sequencing data, with increased coverage. When comparing the two newer kits, we found little difference in cost and workflow, with the NEBNext Ultra both slightly cheaper and faster than the TruSeq Nano. However, the quality of data generated using the TruSeq Nano DNA kit was superior due to higher coverage at regions of low GC content, and more SNPs identified. Researchers should therefore evaluate their resources and the type of application (and hence data quality) being considered when ultimately deciding on which library prep method to use.

Show MeSH
Related in: MedlinePlus