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Scientific Publications on NGS Technologies and General NGS Aspects

This page introduces a selection of scientific publications on general aspects in NGS applications. Most publications are reviews of general interest. 


Publication Introducing Next Generation Sequencing 

Next-Generation Sequencing: Methodology and Application

This paper provides a brief overview of NGS technology and its applications. It describes the differences between whole genome sequencing and targeted sequencing, and is primarily addressed at new users or those with a general interest in NGS.
Grada and Weinbrecht 2013

Scientific Publications on Next Generation Sequencing Technologies & General NGS Aspects

A field guide to whole-genome sequencing, assembly and annotation

This paper introduces a typical whole genome sequencing workflow to new users, in addition to the principles and concepts behind the process. Different challenges, methods, and considerations are also reviewed.
The data analysis workflow is described in terms of the various software tools required for analysis steps such as assembly, quality control, genome annotation, or data management.
In summary, this publication provides a good introduction to the important aspects and considerations in whole genome sequencing.

Ekblom and Wolf 2014

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Assessing the performance of the Oxford Nanopore Technologies MinION

In this study, the MinION NGS system from Oxford Nanopore Technologies was used to re-sequence three bacterial genomes. The results obtained were compared to the reference sequences and to data obtained by Illumina sequencing. In summary, the Nanopore Technologies platform featured a higher sequencing error rate than other NGS technologies, albeit similar to that produced in other published data obtained by using the MinION technology. The error rate depend beside others on the sequencing chemistry. In this study the R6 MinION chemistry was used.
Laver et al. 2015

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Comparison of DNA Quantification Methods for Next Generation Sequencing

6. April 2016, Scientific Reports published the study of Robin et al. "Comparison of DNA Quantification Methods for Next Generation Sequencing"

In this study the utilization of different digital PCR methods (droplet digital PCR (ddPCR) and 5'tailed droplet digital PCR (ddPCR-Tail)) for DNA quantification are compared. The aim of the study is to compare quantification methods and propose solutions that require very small DNA sample amounts for quantification. This will help to reduce unneeded amplification steps that are otherwise only necessary to obtain enough DNA for quantification.
Universal Probe Technologies (UPL®, Roche) was compared with TaqMan PCR and 5′ tail PCR in digital PCR utilizing the QX100® ddPCR system. The internal probe based system gave similar signals compared with the tail system. The read quality score obtained after sequencing was compared for the following different quantification methods (qPCR, QBit, ddPCR and ddPCR-Tail).
In summary, the analysis of different NGS titration methods revealed that ddPCR-based
assays as well as QuBit enable optimal capacity utilization of second generation sequencing.

The publication is free available at Nature

Comparison of eleven methods for genomic DNA extraction suitable for large-scale whole-genome genotyping and long-term DNA banking using blood samples

This paper compares different methods for DNA extraction, including the commercial extraction solutions Nucleospin Blood, Nucleospin Blood L, and Nucleospin Blood XL from Macheri and Nagel, as well as modified versions of these kits. An in-house trizole-based protocol was included, as well as the ChargeSwitch gDNA Tissue Mini kit. Kits were compared for efficiency, cost, and extraction quality for subsequent PCR reactions. One limitation of this study is that it mainly focuses on Macheri and Nagel kits and the ChargeSwitch gDNA Tissue Mini kit, despite numerous other extraction solutions available on the market.
Psifidi et al 2015

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Comparison of Next-Generation Sequencing Systems

This review starts with a brief introduction to sequencing in general and its history, followed by an overview of different NGS technologies such as Roche 454, SOLiD™, Illumina HiSeq, and PGM from Ion Torrent. PacBio and Oxford Nanopore are introduced as third generation sequencing technologies. Additionally, software solutions for the applicable technologies are discussed.
In this paper, hardware components and costs, read length, accuracy, read output, running time, advantages and disadvantages are compared. As the publication dates from 2012, it does not include the latest technological updates.
Different applications of NGS technology are briefly introduced, such as de novo and mate-pair sequencing, whole genome or target-region re-sequencing, sequencing of small RNA, transcriptome sequencing, RNA sequencing, epigenomics, and metagenomics. Finally, the BGI corporation is introduced as a cloud service provider and the world´s largest sequencing facility. The authors of this paper are members of BGI.
Liu et al 2012

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Generation of high-affinity DNA aptamers using an expanded genetic alphabet

This paper evaluates the binding affinity of aptamers containing unnatural nucleotides using the SELEX method. These unnatural nucleotides increase the chemical and structural diversity of aptamers. The binding affinity was subsequently evaluated by using NHS PCR and NGS technology to determine whether the affinity was dependent on the unnatural nucleotides.
Kimoto et al 2013


Library construction for next-generation sequencing: Overviews and challenges

This publication provides a useful introduction to library preparation for different NGS applications, such as library preparation with fragmentation or based library preparation on target specific amplification. The authors describe the different steps of library preparation, including fragmentation, size selection, protocols for RNA depletion, enrichment, and library preparation for RNA and DNA samples. Additionally, protocols for first strand cDNA synthesis are presented. Various considerations such as PCR bias and amplification limitations of GC-rich regions and batch effects are discussed. Furthermore, the targeted approach for sample preparation is discussed, and other methods such as mate pair, CHip, Rip/CLIP and methyl sequencing are introduced.
Head et al. 2014


Non-random DNA fragmentation in next-generation sequencing

The authors of this paper provide an introduction to the NGS workflow along with various reasons for bias during sequencing, such as that which arises during sequencing of GC-rich regions, or NGS bias during DNA fragmentation. The authors examines shearing by sonication, nebulisation and Covaris protocols, and observes a nucleotide sequence-dependent bias in sonication-based shearing. Shearing methods based on hydrodynamic forces (nebulisation and Covaris protocols) also produced bias during fragmentation. In summary, the paper addresses various reasons for bias in NGS.
Poptsova et al. 2014

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Pitfalls of DNA Quantification Using DNA Binding Fluorescent Dyes and Suggested Solutions

3. March 2016, PlosOne published the study of Nakayama et al. "Pitfalls of DNA Quantification Using DNA Binding Fluorescent Dyes and Suggested Solutions".

This study describes the evaluation of three DNA quantification methods for next generation sequencing: NanoDrop 2000 (measures the maximal UV absorbance of nucleic acids including: double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), RNA and nucleotides), Qubit 3.0 (fluorescence spectrometer, measures the fluorescent dye specifically bound to dsDNA), and quantitative PCR (targeting the glyceraldehyde-3-phosphate dehydrogenase, using TaqMan Gene Expression Assays (Life Technologies)).
For evaluation of the different methods DNA was extracted from frozen tissues, formalin-fixed, paraffin-embedded (FFPE) liver tissues, and DNA extracted from the remaining fractions after RNA extraction with Trizol reagent. For testing of DNA samples they were serial diluted in water and different buffers.
Results of measuring frozen DNA showed that Qubit values were significantly lower in highly diluted samples than NanoDrop and qPCR values. It was shown that this was dependent from the salt concentration in the dilution. DNA dilutions in TE buffer and NaCl showed similar values for Qubit and NanoDrop DNA quantification.
For FFPE DNA the Qubit and NanoDrop values differ significant. It is argued that this is due to the degradation of the DNA in the FFPE samples and that Qubit is superior to NanoDrop for quantifying FFPE-DNA, but qPCR is favorable for estimation of the degree of fragmentation in FFPE DNA samples.
For Trizol DNA samples the Qubit value were significant lower than the NanoDrop values. It is argued that this is due to denaturation of dsDNA into ssDNA in Trizol samples.
In conclusion, for DNA measuring using the Qubit method the sample material, salt concentration, and extraction method (buffer e.g. Trizol) need to be considered.

The publication is free available at PlosOne

Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition

In this publication, transposase-catalysed fragmentation is compared with standard NGS library construction protocols. Different protocols based on transposase fragmentation are introduced, including multiplexing 96 bacterial samples, human exome sequencing, shotgun library construction from low input DNA sample, a PCR-free protocol, and single-step preparation of genomic sequencing libraries directly from bacterial colonies.
The coverage rates obtained after fragmentation by physical shearing, enzymatic restriction or transposase based protocols are compared, and the limit of input DNA for library construction from E. coli and human samples was tested.
In summary, transposase-catalysed protocols are faster and less labour-intensive compared to current standard protocols used for library preparation. The method also works with low input sample DNA, and enables multiplexing of barcoded samples and direct sequencing from bacterial colonies without DNA extraction. Transposase-based technology, however, retains bias during library preparation and features limited control over fragment size selection.
Of note, this publication dates from 2010, and numerous library preparation solutions have since become available, along with the optimisation of "standard library prep" methods.
Adey et al. 2010

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Systematic Comparison of Three Methods for Fragmentation of Long-Range PCR Products for Next Generation Sequencing

This paper reviews the most commonly-applied methods for DNA fragmentation ,including nebulisation (performed for one minute with nitrogen at a pressure of 2.1 bar), sonication (Bioruptor™), and enzymatic fragmentation (NEBNext™ dsDNA Fragmentase kit). Enzymatic fragmentation showed the most consistency of the three library preparation methods, but was slightly inferior in performance to sonication and nebulisation with regard to insertions and deletions in the raw sequence reads.
Knierim et al 2011

The Application of Next Generation Sequencing in DNA Methylation Analysis

This review introduces DNA methylation and its corresponding analysis methods. It explains the bisulphite sequencing method and its application in NGS. Different NGS technologies and their application to bisulphite sequencing are described, but as this publication dates from 2010, it does not include the latest NGS technology available. The paper also describes the challenges and requirements for correct data generation, and discussed factors in data misinterpretation such as coverage rate, clonal DNA amplification after bisulphite conversion, and incomplete cytosine conversion. The analysis of huge amounts of NGS data is a further limitation to correct DNA methylation analysis. The author further discusses how new sequencing technologies such as PacBio enable DNA methylation analysis without bisulphite conversion.
Zhang and Jeltsch 2010

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The IGNITE network: a model for genomic medicine implementation and research

This publication describes a study about the investigation and development of practical models for integration of genomic data in electronic health system in order to improve point of care decision making. To facilitate this the IGNITE (Implementing GeNomics In pracTicE; network was established. The aim of the network was to link existing genomic medicine implementation efforts, develop new collaboration projects, and define and share best practice for genomic medicine implementation.
The six projects implemented in this network are introduced and outcome are discussed. The projects are: implementation, adoption and utility of family history in diverse care settings, genetic testing to understand and address renal disease disparities (GUARDD), INGENIOUS: Indiana genomics implementation: an opportunity for the underserved, UF health personalized medicine program, genomic diagnosis and personalized therapy for highly penetrant genetic diabetes, and integrated, individualized and intelligent prescribing (I3P) network.
Weitzel et al. 2016

The publication is free available at NCBI

Ultra-precise detection of mutations by droplet-based amplification of circularized DNA

10. of March 2016, BMCGenomics published the study of Wang et al. "Ultra-precise detection of mutations by droplet-based amplification of circularized DNA"

Circular sequencing (CirSeq) enables the replicated sequencing of single strand circularized molecules by rolling circle amplification (RCA). The study introduces the new Droplet-CirSeq method. This method combines millions of pico-liter droplets with circular sequencing. Droplet-CirSeq reduces the amplification bias compared to Cir-Seq und enables the more effective detection of rare mutations, Droplet-CirSeq enables an error rate of 3 ~ 5 X 10-6.
In this method the DNA is fragmentized in small fragment and afterward circularized. Subsequently, the circularized DNA fragments are amplified in droplet digital PCR. The resulting amplified DNA is fragmented and used for standard library preparation.
In order to eliminate PCR and sequencing errors the initial DNA fragments used for circularization are less than half as long as the read length of the final sequencing reads, this ensures repeated sequencing of these fragments.
The sensitivity, efficiency, base error rates and error pattern of Droplet-CirSeq were compared to standard CirSeq. The false positive and false negative rate for detection of SNPs were evaluated for both methods with different DNA sample amount input. As samples two E. coli strains were mixed with an 1:10 ration and investigated with both methods.
In conclusion, Droplet CirSeq shows a more uniform amplification, more accurate mutation detection frequency, and lower false positive and false negative rates compared to standard CirSeq.

The publication is free available at BMC Genomics

Updating benchtop sequencing performance comparison

In this publication, the three most commonly-used NGS technologies (Ion Torrent PGM, Illumina My Seq, and Roche 454) are compared by sequencing the enterohemorrhagic E. coli O157:H7 Sakai strain. The sequencing was performed at different institutes, using the chemistry recommended by the technology provider. Assembling of data with different coverage rates are compared, and differences between insertion/deletion errors and substitutions between the three NGS technologies are contrasted to the reference Sanger-sequenced data sets. Significant differences between insertion/deletion errors rates were observed.
Jünemann et al. 2013


Scientific Publications on NGS Applications in Vaccine Research

Deep Sequencing in Pre- and Clinical Vaccine Research

In this review, a brief introduction to NGS technologies is followed by a discussion of the various fields of NGS application in vaccine research and development, including reverse vaccinology, deep sequencing of highly variable antigenic regions, development of anti-cancer vaccines, and post-marketing monitoring of vaccine coverage. The use of NGS in the generation of safer vaccines, such as by vaccine lot release control during production, is discussed. In summary, this review suggests that vaccine research and production can broadly benefit from future advances in NGS technology in order to generate safer and more effective vaccines.
Prachi et al. 2013

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