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Effects and considerations of multiplexing ForenSeq Kintelligence libraries with a negative control.

Gunmeet Kaur Bali¹, Daniela Cuenca¹, Jeanette Wallin¹

¹California Department of Justice, Jan Bashinski DNA Laboratory, Richmond, California, USA.

Electrophoresis

|March 7, 2024

Summary

Including a negative control in every forensic massively parallel sequencing (MPS) run consumes valuable sequencing space, potentially causing allele dropout and increasing unknown associations in genetic genealogy databases.

Keywords:

GEDmatch PRO Kintelligence kinship associations negative control sequence reads

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Area of Science:

Forensic Science
Genetics
Molecular Biology

Background:

Negative controls are crucial for monitoring contamination in forensic DNA analysis.
Massively parallel sequencing (MPS) is increasingly adopted in forensic laboratories.
Current FBI Quality Assurance Standards mandate negative controls in forensic DNA analysis.

Purpose of the Study:

To evaluate the necessity and impact of including a negative control in every sequencing run using the ForenSeq Kintelligence Kit.
To assess the utility of negative controls in subsequent sequencing runs after initial library preparation.

Main Methods:

Investigated the effect of including a negative amplification control in each ForenSeq Kintelligence sequencing run.
Quantified the sequencing space occupied by the negative control library.
Analyzed the impact on DNA-containing samples, including allele/locus dropout and unknown associations in GEDmatch PRO.

Main Results:

Negative control libraries occupied 7%-14% of the total sequencing run output.
Reduced sequencing space for DNA samples led to allele or locus dropout in some cases.
Increased instances of sixth to seventh order unknown associations were observed in GEDmatch PRO.

Conclusions:

The routine inclusion of negative controls in every sequencing run of the ForenSeq Kintelligence Kit is not essential after initial library preparation.
The practice consumes significant sequencing resources, potentially compromising the analysis of valuable forensic DNA samples.
Optimizing negative control usage can improve MPS efficiency and data quality in forensic genetic genealogy.