Optimization of Illumina® Nextera® XT Library Preparation for Analysis of Complete Mitochondrial Genomes from Human Reference Samples

Abstract Background: Optimized and efficient library preparation workflow is one of the most important prerequisites for obtaining high quality and quantity of results in massively parallel sequencing (MPS). Our aim was to assess and optimize different steps of Illumina® Nextera® XT assay for analysis of whole mitochondrial genomes.Methods and Results: Among the three long-range high-fidelity DNA polymerases tested here, PrimeSTAR® GXL performed best in aspects of specificity and yield for mitochondrial DNA (mtDNA) enrichment. Furthermore, library quantification combined with individual library-by-library dilution outperformed bead-based normalization in terms of more equal distribution of reads per library, reduced hands-on time and simplified workflow. Increasing the number of amplification cycles in the index-adapters-adding PCR step had no adverse effect on the level of sequencing noise, which remained low both in negative controls and in samples.Conclusions: Optimizations described herein provide beneficial insights for laboratories aiming at implementation and/or advancement of similar MPS workflows (e.g. small genomes, PCR amplicons and plasmids).

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Massively parallel sequencing of complete mitochondrial genomes from hair shaft samples

Forensic Science International Genetics ◽

10.1016/j.fsigen.2014.11.009 ◽

2015 ◽

Vol 15 ◽

pp. 8-15 ◽

Cited By ~ 56

Author(s):

Walther Parson ◽

Gabriela Huber ◽

Lilliana Moreno ◽

Maria-Bernadette Madel ◽

Michael D. Brandhagen ◽

...

Keyword(s):

Massively Parallel Sequencing ◽

Hair Shaft ◽

Massively Parallel ◽

Mitochondrial Genomes ◽

Parallel Sequencing ◽

Complete Mitochondrial Genomes

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High polymorphism detected by massively parallel sequencing of autosomal STRs using old blood samples from a Chinese Han population

Scientific Reports ◽

10.1038/s41598-019-55282-9 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Wenshen Dai ◽

Yajiao Pan ◽

Xiaochen Sun ◽

Riga Wu ◽

Luo Li ◽

...

Keyword(s):

Massively Parallel Sequencing ◽

Chinese Han Population ◽

Massively Parallel ◽

Library Preparation ◽

Chinese Han ◽

Discrimination Power ◽

Blood Samples ◽

Parallel Sequencing ◽

Han Population ◽

Str Loci

AbstractThe development of massively parallel sequencing (MPS) has quickly changed forensic short tandem repeat (STR) genotyping. By providing detailed sequence information, MPS technology may be used as an alternative or additional method to overcome the limitations of capillary electrophoresis-based STR profiling. Most current NGS processes are labour-intensive with regard to library preparation and require high-quality DNA template. In this study, a 16-plex STR typing system (SeqType®R16) was used to achieve direct library preparation without DNA extraction and adaptor ligation. The efficiency of this system was tested in 601 individuals, including 593 old blood samples from the Chinese Han population and eight positive controls. It took approximately 4 hours for library preparation, including blood direct multiplex PCR (1.5 hours), mixing of the product (15 minutes), single tube purification (2 hours) and quantification (15 minutes). The results showed that MPS presented a broader allele range and higher discrimination power. Except for FGA and D19S433, the allele number almost doubled or more than doubled at all complex STR loci and simple STR loci, including D13S317, D16S539, D5S818, and D7S820. The range of discrimination power increased from 0.8008–0.9572 to 0.8401–0.9753, and the culminated matching probability decreased from 1.7 × 10−15 to 1.1 × 10−17.

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Comparison of whole mitochondrial genome variants between hair shafts and reference samples using massively parallel sequencing

International Journal of Legal Medicine ◽

10.1007/s00414-019-02205-y ◽

2019 ◽

Vol 134 (3) ◽

pp. 853-861

Author(s):

Bo Min Kim ◽

Sae Rom Hong ◽

Hein Chun ◽

Sangwoo Kim ◽

Kyoung-Jin Shin

Keyword(s):

Mitochondrial Genome ◽

Massively Parallel Sequencing ◽

Massively Parallel ◽

Parallel Sequencing ◽

Hair Shafts ◽

Reference Samples ◽

Genome Variants

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Advanced Whole Genome Sequencing Using an Entirely PCR-free Massively Parallel Sequencing Workflow

10.1101/2019.12.20.885517 ◽

2019 ◽

Cited By ~ 1

Author(s):

Hanjie Shen ◽

Pengjuan Liu ◽

Zhanqing Li ◽

Fang Chen ◽

Hui Jiang ◽

...

Keyword(s):

Machine Learning ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Massively Parallel Sequencing ◽

Variant Calling ◽

Systematic Errors ◽

Massively Parallel ◽

Whole Genome ◽

Library Preparation ◽

Parallel Sequencing

AbstractBackgroundSystematic errors can be introduced from DNA amplification during massively parallel sequencing (MPS) library preparation and sequencing array formation. Polymerase chain reaction (PCR)-free genomic library preparation methods were previously shown to improve whole genome sequencing (WGS) quality on the Illumina platform, especially in calling insertions and deletions (InDels). We hypothesized that substantial InDel errors continue to be introduced by the remaining PCR step of DNA cluster generation. In addition to library preparation and sequencing, data analysis methods are also important for the accuracy of the output data.In recent years, several machine learning variant calling pipelines have emerged, which can correct the systematic errors from MPS and improve the data performance of variant calling.ResultsHere, PCR-free libraries were sequenced on the PCR-free DNBSEQ™ arrays from MGI Tech Co., Ltd. (referred to as MGI) to accomplish the first true PCR-free WGS which the whole process is truly not only PCR-free during library preparation but also PCR-free during sequencing. We demonstrated that PCR-based WGS libraries have significantly (about 5 times) more InDel errors than PCR-free libraries.Furthermore, PCR-free WGS libraries sequenced on the PCR-free DNBSEQ™ platform have up to 55% less InDel errors compared to the NovaSeq platform, confirming that DNA clusters contain PCR-generated errors.In addition, low coverage bias and less than 1% read duplication rate was reproducibly obtained in DNBSEQ™ PCR-free using either ultrasonic or enzymatic DNA fragmentation MGI kits combined with MGISEQ-2000. Meanwhile, variant calling performance (single-nucleotide polymorphisms (SNPs) F-score>99.94%, InDels F-score>99.6%) exceeded widely accepted standards using machine learning (ML) methods (DeepVariant or DNAscope).ConclusionsEnabled by the new PCR-free library preparation kits, ultra high-thoughput PCR-free sequencers and ML-based variant calling, true PCR-free DNBSEQ™ WGS provides a powerful solution for improving WGS accuracy while reducing cost and analysis time, thus facilitating future precision medicine, cohort studies, and large population genome projects.

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