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Library construction optimization and analysis of Short Tandem Repeats by a simple, PCR-based DNA barcoding method

Soma, Markus André LU (2020) KMBM01 20201
Applied Microbiology
Biotechnology
Abstract
DNA profiling is evolving in the forensics community towards introducing massively parallel sequencing (MPS) as a complement to capillary electrophoresis (CE). Obstacles remain however before this technology can become routine in forensic casework, such as the development of more efficient bioinformatics solutions and recommendations concerning data analyses. Additionally, there is a need to develop MPS methods that are even more sensitive than current commercialized systems. One promising candidate is SiMSen-Seq, a method that incorporates unique molecular identifiers (UMI’s), also known as barcodes, into PCR library preparation, allowing for the reduction of background noise (artefacts) in data analyses. In the development of SiMSen-Seq... (More)
DNA profiling is evolving in the forensics community towards introducing massively parallel sequencing (MPS) as a complement to capillary electrophoresis (CE). Obstacles remain however before this technology can become routine in forensic casework, such as the development of more efficient bioinformatics solutions and recommendations concerning data analyses. Additionally, there is a need to develop MPS methods that are even more sensitive than current commercialized systems. One promising candidate is SiMSen-Seq, a method that incorporates unique molecular identifiers (UMI’s), also known as barcodes, into PCR library preparation, allowing for the reduction of background noise (artefacts) in data analyses. In the development of SiMSen-Seq towards its use in forensics, the library preparation protocol, consisting of two distinct PCRs (PCR1 and PCR2), were in this project further optimized for the efficient amplification of short tandem repeats (STRs). By applying Bioanalyzer 2100, the results showed that the type of DNA polymerase and the barcode primer concentration had the greatest effect in maximizing specific products and minimizing nonspecific products. SuperFi and Immolase, two promising DNA polymerases that resulted in efficient STR amplification, were further evaluated for use in library preparation by MPS using MiSeq, and the results showed that although the use of SuperFi in PCR1 and Immolase in PCR2 resulted in the most STR products, it generated the highest amount of artefacts, complicating data interpretation. Instead, utilizing SuperFi, a proofreading enzyme, in PCR2 of library preparation, decreased the amount of generated artefacts. Based on these results, it is therefore recommended that further tests are performed with SuperFi in both PCR1&2 of library preparation. Testing other DNA polymerase combinations featuring proofreading abilities may also provide valuable data. However, before SiMSen-Seq can be implemented in the analyses of real crime scene samples, additional evaluation using low DNA concentrations and more complex DNA samples, including inhibitors, is required. Further customization of the bioinformatic data workflow is also necessary to streamline the work process. (Less)
Popular Abstract
Forensic DNA analyses of complex crime scene samples can be improved using an optimized method

Optimization of the method led to increased production of DNA that is necessary to correctly identify and distinguish one person from another. DNA errors were reduced, and interestingly, changing one factor of the method had the power to further reduce DNA errors, simplifying interpretation of a DNA sample.

The Swedish National Forensic Centre (NFC) handles thousands of crime scene samples every year, many of which are highly complex or contain very little evidence in the form of DNA, complicating the work for forensic scientists. In this project, the focus is on the optimization of a method that may one day lead to improved analysis of... (More)
Forensic DNA analyses of complex crime scene samples can be improved using an optimized method

Optimization of the method led to increased production of DNA that is necessary to correctly identify and distinguish one person from another. DNA errors were reduced, and interestingly, changing one factor of the method had the power to further reduce DNA errors, simplifying interpretation of a DNA sample.

The Swedish National Forensic Centre (NFC) handles thousands of crime scene samples every year, many of which are highly complex or contain very little evidence in the form of DNA, complicating the work for forensic scientists. In this project, the focus is on the optimization of a method that may one day lead to improved analysis of crime scene DNA evidence, such that in the future, it will be easier to connect perpetrators with crimes.

When a method is being adapted from its use in one field to another, in this case from cancer diagnostics to forensic analyses, optimization under controlled conditions (i.e. in the laboratory) is an important part of the development process. By changing different factors of the method, it can be fine-tuned towards providing better quality data, making analyses easier. Although these factors can be difficult and time-consuming to discover, it is an important job towards implementing a new method in a laboratory handling real samples.

One key factor determined in this project was the type of DNA polymerase used when running the method. The DNA polymerase can be thought of as a machine that is necessary in building more DNA from the very small amounts of DNA that is found at the crime scene (e.g. in blood or saliva), enabling identification of who left the stain. An additional important factor determined in this optimization was the primer concentration. The primer specifies what part of the DNA that should be built, such that the identity of an individual can be correctly determined and distinguished from another person’s DNA (as the DNA differs between individuals). The essence of the method is that something that can be thought of as a barcode will be added to all of the newly built DNA, which allows for a smoother process in identifying the person(s) who committed the crime.

After testing many different types of DNA polymerases, as well as various primer concentrations and other factors of the method, one specific DNA polymerase at a set primer concentration led to the production of less DNA errors, making it easier to identify the correct person(s) in the sample. DNA errors often occur when building new DNA fragments, and some DNA polymerases produce more and others less, which was shown in this study. It is therefore important to use this specific DNA polymerase further or test other low error polymerases when running this method. Errors in the produced DNA could also be reduced when taking advantage of the barcodes, simplifying data interpretation. It was then also possible to successfully distinguish two different persons from each other based on differences in their DNA. Although no real crime samples were analyzed in this project, the results look very promising.

Towards the future development of this method, more testing should be done with the goal to further reduce the amount of errors produced in the DNA when using the method. Additional customization of the analyses workflow when handling computer software is also required to further advance the method in reaching its full potential, such that it may one day be used to analyze real crime scene samples. (Less)
Please use this url to cite or link to this publication:
author
Soma, Markus André LU
supervisor
organization
alternative title
Optimering av bibliotekskonstruktion och analys av Short Tandem Repeats med en enkel PCR-baserad DNA barcoding metod
course
KMBM01 20201
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Forensics, DNA, Short Tandem Repeats, Unique Molecular Identifiers, Polymerase Chain Reaction, Capillary Electrophoresis, Massively Parallel Sequencing, Applied Microbiology, Teknisk mikrobiologi
language
English
id
9016965
date added to LUP
2020-06-30 16:11:56
date last changed
2020-07-02 10:00:47
@misc{9016965,
  abstract     = {DNA profiling is evolving in the forensics community towards introducing massively parallel sequencing (MPS) as a complement to capillary electrophoresis (CE). Obstacles remain however before this technology can become routine in forensic casework, such as the development of more efficient bioinformatics solutions and recommendations concerning data analyses. Additionally, there is a need to develop MPS methods that are even more sensitive than current commercialized systems. One promising candidate is SiMSen-Seq, a method that incorporates unique molecular identifiers (UMI’s), also known as barcodes, into PCR library preparation, allowing for the reduction of background noise (artefacts) in data analyses. In the development of SiMSen-Seq towards its use in forensics, the library preparation protocol, consisting of two distinct PCRs (PCR1 and PCR2), were in this project further optimized for the efficient amplification of short tandem repeats (STRs). By applying Bioanalyzer 2100, the results showed that the type of DNA polymerase and the barcode primer concentration had the greatest effect in maximizing specific products and minimizing nonspecific products. SuperFi and Immolase, two promising DNA polymerases that resulted in efficient STR amplification, were further evaluated for use in library preparation by MPS using MiSeq, and the results showed that although the use of SuperFi in PCR1 and Immolase in PCR2 resulted in the most STR products, it generated the highest amount of artefacts, complicating data interpretation. Instead, utilizing SuperFi, a proofreading enzyme, in PCR2 of library preparation, decreased the amount of generated artefacts. Based on these results, it is therefore recommended that further tests are performed with SuperFi in both PCR1&2 of library preparation. Testing other DNA polymerase combinations featuring proofreading abilities may also provide valuable data. However, before SiMSen-Seq can be implemented in the analyses of real crime scene samples, additional evaluation using low DNA concentrations and more complex DNA samples, including inhibitors, is required. Further customization of the bioinformatic data workflow is also necessary to streamline the work process.},
  author       = {Soma, Markus André},
  keyword      = {Forensics,DNA,Short Tandem Repeats,Unique Molecular Identifiers,Polymerase Chain Reaction,Capillary Electrophoresis,Massively Parallel Sequencing,Applied Microbiology,Teknisk mikrobiologi},
  language     = {eng},
  note         = {Student Paper},
  title        = {Library construction optimization and analysis of Short Tandem Repeats by a simple, PCR-based DNA barcoding method},
  year         = {2020},
}