Development and Evaluation of the Ancestry Informative Marker Panel of the VISAGE Basic Tool

Round 1

Reviewer 1 Report

The topic of the manuscript Development and evaluation of the ancestry informative marker panel of the VISAGE Basic Tool is of great interest for the forensic community and a current overview over the existing studies might be helpful in moving it forward.
This is a very well written manuscript and correct in the methodology of the research.

The paper is very thorough and includes a relevant step on the topic of ancestry study by SNPs. The exploration of VISAGE Basic Tool is very interesting.

Author Response

We would like to thank the Reviewer for the time they dedicated to scrutiny of the manuscript and their positive feedback. We knew the paper was quite data-dense prior to submission and we greatly appreciate the careful checks made to ensure it was appropriately prepared for publication.

Reviewer 2 Report

It is considered a very informative and enterprising thesis.

Author Response

We would like to thank the Reviewer for the time they dedicated to scrutiny of the manuscript and their positive feedback. We knew the paper was quite data-dense prior to submission and we greatly appreciate the careful checks made to ensure it was appropriately prepared for publication.

Reviewer 3 Report

I read with interest this manuscript submitted to the magazine "Genes". This is a careful analysis of the possibilities offered by new technologies in the study, classification and use for diagnostic purposes of single nucleotide polymorphisms and more. I find that the "material and methods" part is well structured, such as results and discussion. I just suggest you expand on the conclusions and do a light style of English.

Author Response

We would like to thank the Reviewer for the time they dedicated to scrutiny of the manuscript and their positive feedback. We knew the paper was quite data-dense prior to submission and we greatly appreciate the careful checks made to ensure it was appropriately prepared for publication.

We have redrafted the 'Concluding remarks' section to simplify the text and clarify some long passages. This section has also been expanded to include further points of discussion not covered in the previous section.

Revised text:

4. Concluding remarks

The primary aim of the VISAGE Consortium of creating an all-in-one forensic intelligence tool based on MPS technology, requires the development of a full analytical pipeline. This should include population reference data and predictive algorithms for the inference of age, ancestry and appearance characteristics associated with the SNPs genotyped, plus an optimum sequencing protocol applicable to the two main forensic MPS platforms in current use. The strategy to start with a prototype tool of simple age predictors in one multiplex, and established pigmentation phenotype and continental ancestry predictors in the second, has allowed us to build a strong foundation on which to develop the more ambitious VISAGE Enhanced Tool (ET). ET represents substantial expansion of all three ‘AAA’ predictive systems in the same parallel MPS multiplex set-up as BT (i.e., separate DNA workflows dedicated to methylation analysis and SNP genotyping). The ancestry panel of BT therefore took the best ancestry informative SNPs from several well-established forensic ancestry panels, with particular regard to known MPS sequence reliability as well as optimum ancestry informativeness. For this reason, BT has provided very good sequencing performance with forensic DNA [29,38] and enables ancestry inferences to be made for continental and South Asian populations of origin with the highest levels of statistical likelihood.

Although we dedicated considerable resources to genotyping BT SNPs in populations addressing several geographic gaps in coverage left by large-scale genomic sequencing projects, many global regions remain to be characterised with new studies. The dynamic system in Snipper of compiling the largest possible array of population data and allowing the end-user to select which samples extend or modify the standardised reference set, provides flexibility for statistical analysis of BT SNP data. It also allows new population data to be uploaded to the webpages holding reference sets (http://mathgene.usc.es/snipper/forensic_mps_aims.html) and the additional populations shown in File S1. Although genetic cluster analysis using STRUCTURE can be challenging for forensic practitioners analysing population data for the first time, in our view it is the best way to detect and analyse individual co-ancestry in persons with admixed backgrounds. The comparisons of GDA and STRUCTURE made in this study indicate GDA is not accurate enough to evaluate low level co-ancestry when admixture patterns are complex and an individual has more than two co-ancestries. However, when the BT ancestry SNPs are compared to the much larger panel of Human Origins SNPs, the level of detail obtained using STRUCTURE is well matched and indicates BT SNPs are informative enough to accurately detect co-ancestral components present above 10% proportions.

Lastly, the care taken in selecting the BT ancestry SNPs based on their recorded reliability and sensitivity in previous MPS-based forensic ancestry panels [8,12,18,20,23] has helped to ensure predictable performance going forward with evaluation of both forensic MPS platforms. This extends to the sequencing chemistry, library preparation and sequence read balance (between strands; between alleles, within loci; between loci) observed so far in the forensic validations the VISAGE Consortium has made following SWGDAM guidelines for newly developed forensic DNA techniques (AmpliSeq chemistry [29], PowerSeq chemistry [38], and ForenSeq chemistry with a manuscript in submission). These same principals have been applied to the development of the ancestry panel of ET and has provided the means to increase this tool’s overall multiplex size and extend the range of populations the ET ancestry SNPs can differentiate.