DNA testing

There are two sources of reference deoxyribonucleic acid (DNA) samples that may be collected for the investigation of unidentified and missing persons:

•    A direct reference sample is a DNA sample collected from a missing person’s stored medical specimens (e.g. newborn screening card, blood sample, biopsy sample) or personal items (e.g. toothbrush, razor, hairbrush). The missing person’s DNA is extracted from these biological samples for comparison to unidentified human remains (UHR) DNA profiles for DNA matching purposes. Direct reference samples are typically obtained with the permission of the relative(s) of the missing person (where available).

•    A family reference sample is a DNA sample collected from suitable genetic relatives of the missing person. The DNA is typically obtained from a self-administered mouth swab that removes cells from the inner cheek. The relative’s DNA is extracted from these cells for comparison to UHR DNA profiles for DNA matching purposes. Family reference samples are always obtained with the informed consent of the relative(s) of the missing person volunteering the sample or their parent/guardian if under 18 years of age.
 

Direct and/or family reference samples should be collected and tested for every long-term missing person (LTMP). In general, direct reference samples are preferred for deoxyribonucleic acid (DNA) matching purposes over family reference samples (where available) because more simplified statistics can be applied, a higher probability of identity can be achieved and legislation permits them to be readily searched against unidentified human remains (UHR) DNA profiles on the national DNA database.

Direct reference samples:

Stored medical specimens are generally preferred over personal items (where available) as personal items often produce mixed DNA profiles, or there can be uncertainty as to ownership. Newborn screening cards are considered the ‘gold standard’ direct reference sample (where available); however, jurisdictions do have differing legislation prescribing their retention period and secondary uses. These samples can also be important in other scenarios, such as when an adopted child goes missing and no biological parents are known or available.

If personal items are the only direct reference sample available, it is recommended that multiple personal items are collected and tested to confirm a consistent DNA profile is being produced, or family reference samples are also collected and tested to confirm the direct reference sample is attributable to the missing person (where available). Importantly, for historical missing persons cases, neither medical specimens nor personal items may be available and the collection and testing of family reference samples will be essential.

Family reference samples:

Samples from at least two genetic relatives should be collected, with at least one relative having a direct genetic link to the missing person (where available) i.e. a first degree relative such as a parent, child or full sibling. Ideally, one sample should be collected from a close maternal relative (e.g. mother) and the other should be a close paternal relative (e.g. father) (where available).
In order of preference, the following combinations of genetic relatives are generally preferred (where available):
1.    Identical twin
2.    Both parents
3.    Parent, child(ren) plus spouse (or parent-in-common)
4.    Child(ren) plus spouse (or parent-in-common)
5.    Parent and full sibling
6.    Multiple full siblings

For historical missing persons cases, samples from distant maternal or paternal relatives may only be available i.e. a second degree relative such as a grandparent, grandchild, aunt/uncle, niece/nephew or half-sibling, and/or a third degree relative such as a great-grandparent, great-grandchild, great-aunt/uncle or first cousin. In these cases, family reference samples should be collected from all available genetic relatives.
 

The process of collecting a family reference sample is outlined below, and will typically be facilitated by the family’s Case Officer, or the Missing Persons Unit in their state or territory:

1. A family member gives informed consent in the presence of an independent person to provide a voluntary reference deoxyribonucleic acid (DNA) sample for the purposes of assisting to identify a missing or deceased person. Note: A family member may withdraw consent to the forensic procedure being performed at any time.
2. A family member can obtain legal advice before deciding whether or not to consent to the forensic procedure.
3. A police officer will supervise the collection of a reference DNA sample using a non-intimate forensic procedure; this is usually in the form of a self-administered mouth swab. 
4. The forensic procedure may be recorded by electronic (audio and/or visual) means.
5. A police officer will confirm and document the genetic relationship of the family member to the missing person.
6. After collection, the reference DNA sample will be packaged securely and transported to the forensic laboratory for DNA testing.

To identify unidentified human remains (UHR), the deoxyribonucleic acid (DNA) profile recovered from the UHR must be matched to a nominated DNA profile from the long-term missing person (LTMP) or their genetic relative(s). Alternatively, a secure, national law enforcement database of DNA profiles from missing persons, missing persons’ relatives and other relevant categories can be searched to find DNA matches.

If direct DNA searching is used, DNA from a direct reference sample may be a direct match with DNA from UHR. This is possible because an individual’s DNA is the same in every cell of the body. In the case of such a match, the identity of the UHR can be confirmed.

If familial/kinship DNA searching is used, DNA from a family reference sample may partially match with DNA from UHR. This is possible because genetic relatives share similar and predictable patterns in their DNA profiles. The closer the genetic relationship between the family reference sample and the UHR, the more DNA they share. In the case of such a partial match, the identity of UHR may be inferred. Additionally, other kinds of DNA testing can be performed to support this genetic relationship between the UHR and putative relative(s).
 

There are different kinds of deoxyribonucleic acid (DNA) found in most cells of the body and they have different inheritance patterns. The presence (or absence) of these different kinds of DNA in unidentified human remains (UHR) will determine the type of DNA testing that is able to be performed, and therefore the family reference samples able to be used for DNA matching purposes.

Nuclear DNA

Nuclear DNA is located in a compartment of the cell called the nucleus. It includes DNA contained in both autosomal and sex chromosomes.

DNA inherited from autosomal chromosomes

There are 22 pairs of autosomal chromosomes. One chromosome of each pair is inherited from one biological parent and the other chromosome is inherited from the other biological parent. The DNA from the biological parents is shuffled in a process called recombination so that siblings may have different DNA segments on the chromosomes inherited from each biological parent.

Autosomal DNA can be used to infer both close and distant genetic relationships. Closely related genetic relatives, such as parents and children or full siblings, will share more and longer DNA segments than more distant genetic relatives, such as grandparents and grandchildren or cousins. Autosomal DNA can also be used to estimate the genetic ancestry and phenotype (i.e. physical appearance) of the UHR, including hair and eye colour.

DNA inherited from sex chromosomes

There are two sex chromosomes called X and Y. They are responsible for sex determination in humans. DNA contained in the X and Y chromosome can be used to infer genetic relationships in specific long-term missing persons (LTMP) cases.

X chromosomes

Females have two X chromosomes. One X chromosome is inherited from one biological parent and the other X chromosome is inherited from the other biological parent. As for autosomal DNA, the pair of X chromosomes undergo recombination so that females pass a different X chromosome to each of their offspring.

Males have one X chromosome, inherited from their biological mother only. They pass this X chromosome on to their female offspring whole, without recombination.

Because of this unique, sex-specific mode of inheritance, X chromosome DNA can be used to infer genetic relationships that can be difficult to establish from autosomal DNA alone, such as half-siblings who only have one biological parent in common.

Y chromosomes

Males have one Y chromosome, inherited whole from their biological father, without recombination. Females do not have a Y chromosome.

Because of this unique, sex-specific mode of inheritance, Y chromosome DNA can be used to infer paternal lineage, such as lineage shared by a father and his son or brothers. Since all males in a paternal line are expected to have the same Y chromosome DNA, close or distant paternal relatives can provide suitable family reference samples for Y chromosome DNA testing.

Mitochondrial DNA

Mitochondrial DNA is located in a compartment of the cell called the mitochondrion. There are multiple mitochondria in each cell, each with multiple copies of mitochondrial DNA. This means that while there are only two copies of autosomal DNA in every cell, there are many more copies of mitochondrial DNA.

Mitochondrial DNA is inherited whole from biological mother to her offspring, without recombination. Because of this unique, sex-specific mode of inheritance, mitochondrial DNA can be used to infer maternal lineage, such as lineage shared by siblings, by a mother and her daughter, or by a mother and her son. Since all individuals in a maternal line are expected to have the same mitochondrial DNA, close or distant maternal relatives can provide suitable family reference samples for mitochondrial DNA testing.
 

There are a number of genetic targets that can be tested from deoxyribonucleic acid (DNA). DNA from unidentified human remains (UHR), direct reference samples and family reference samples will generally be tested in the order shown below, from least information revealed about them to most information, until the identity of UHR can be inferred.

The information relevant to unidentified and missing persons investigations may include:
•    Biological sex
•    Genetic relationships with other individuals
•    Genetic ancestry (UHR only)
•    Certain physical traits e.g. hair and eye colour (UHR only)

The types of genetic targets analysed to estimate this information include:

Short tandem repeats (STRs)

STRs are short, repetitive sequences of DNA that are highly variable amongst humans and can therefore be used to differentiate between them. They can be obtained from autosomal DNA, X chromosome DNA and Y chromosome DNA. There are no STRs in mitochondrial DNA.

The vast majority of DNA profiles generated in forensic laboratories target STRs. These profiles are typically used to match two DNA samples from the same source but can also be used to infer close genetic relationships. The number of STR targets in a DNA profile typically varies from 20 to 30. STR DNA profiles also contain one or more genetic targets to estimate biological sex.

Single nucleotide polymorphisms (SNPs)

SNPs are single differences in the DNA sequence. They can be obtained from autosomal DNA, X chromosome DNA, Y chromosome DNA and mitochondrial DNA. The number of targets in a SNP DNA profile can vary from 50 to one million.

Like STRs, SNPs can be used to match two DNA profiles from the same source. Autosomal SNPs can also be used to infer close genetic relationships (e.g. parent/child, sibling/sibling) and more distant genetic relationships as the number of SNPs analysed increases (e.g. third cousins or further). They can also be used to estimate an individual’s genetic ancestry and phenotype (i.e. physical appearance). Autosomal SNPs can be categorised according to their predictive function:

•    Identity informative SNPs
•    Kinship informative SNPs
•    Ancestry informative SNPs
•    Phenotype informative SNPs

Sequencing of SNPs will be performed in-house by the National DNA Program for Unidentified and Missing Persons to estimate an individual’s identity, close and distant genetic relationships (including for the purpose of forensic investigative genetic genealogy), genetic ancestry, and hair and eye colour. X and Y chromosome SNPs can also be used to estimate biological sex.

Whole genomes

Whole genomes are the complete DNA sequence of the autosomal and sex chromosomes and mitochondria. By sequencing whole genomes, all genetic information about the UHR is obtained, including both STRs and SNPs in autosomal DNA, X chromosome DNA and Y chromosome DNA, and SNPs in mitochondrial DNA.

Sequencing of whole mitochondrial genomes will be performed in-house by the National DNA Program for Unidentified and Missing Persons to determine an individual’s mitochondrial SNP profile for DNA matching purposes.

Sequencing of whole autosomal and sex chromosomes, and mitochondrial genomes, will be performed by the overseas (USA-based) forensic genomics service provider, Othram, to extract the relevant genetic information from the autosomal chromosomes, X chromosome, Y chromosome and mitochondrial genome for the specific purpose of forensic investigative genetic genealogy.
 

The deoxyribonucleic acid (DNA) profiles from direct reference samples will be subject to a number of genetic analyses to aid the process of identifying unidentified human remains (UHR). Initially, they will be used for direct DNA searching and matching using autosomal short tandem repeats (STRs). They may also be used for other types of complementary DNA testing using alternative genetic targets such as direct DNA searching and matching using Y chromosome STRs, mitochondrial single nucleotide polymorphisms (SNPs) and identity informative SNPs.

DNA profiles from direct reference samples can be compared to equivalent DNA profiles from UHR on a case-by-case basis or they may be submitted to external databases for direct DNA searching against a collection of known DNA profiles. Database searches will generally be performed in the order shown below, from least information revealed from the submitted DNA profile to most information, until the identity of the UHR can be inferred.

DNA profiles submitted to law enforcement DNA databases are not publicly available and are typically regularly searched and retained on the database until the LTMP has been located or identified.

1. National Criminal Investigation DNA Database (NCIDD)

The NCIDD is a national DNA database maintained by the Australian Criminal Intelligence Commission (ACIC) for conducting national searches of autosomal STR profiles to link UHR and long-term missing persons (LTMP).

2. NCIDD-Integrated Forensic Analysis (NIFA)

The NIFA is a national DNA database maintained by the ACIC for conducting national searches of Y chromosome STR and mitochondrial SNP profiles to link UHR and LTMP.

3. International Criminal Police Organisation (INTERPOL) DNA Database

The INTERPOL DNA Database is an international DNA database maintained by INTERPOL for conducting international searches of autosomal STR profiles to link UHR and LTMP.

4. Other International Law Enforcement DNA Databases

National law enforcement DNA databases in other countries may also be used for conducting international searches of autosomal STR profiles, Y chromosome STR and mitochondrial SNP profiles to link UHR and relatives of LTMP.

National law enforcement DNA databases in other countries may also be used for conducting international searches of autosomal STR profiles, Y chromosome STR and mitochondrial SNP profiles to link UHR and relatives of LTMP.

The deoxyribonucleic acid (DNA) profiles from family reference samples will be subject to a number of genetic analyses to aid the process of identifying unidentified human remains (UHR). These include:

• Short range familial/kinship searching and matching to identify close genetic relatives using one or more genetic targets such as autosomal short tandem repeats (STRs), Y chromosome STRs and single nucleotide polymorphisms (SNPs), mitochondrial SNPs, X chromosome STRs and SNPs, and kinship informative SNPs
• Medium to long range familial/kinship searching and matching to identify more distant genetic relatives using kinship informative SNPs

DNA profiles from family reference samples can be compared to equivalent DNA profiles from UHR on a case-by-case basis or they may be submitted to external databases for familial/kinship searching against a collection of known DNA profiles. Where practicable, consent will be sought from genetic relatives who provide family reference samples to have their DNA profiles submitted to external databases, when direct reference samples fail to identify the UHR or are not available. They will generally be utilised in the order shown below, from least information revealed from the submitted DNA profile to most information, until the identity of the UHR can be inferred.

DNA profiles submitted to law enforcement DNA databases are not publicly available and are typically regularly searched and retained on the database until the long-term missing person (LTMP) has been located or identified, or the genetic relative who voluntarily provided the family reference sample requests that it be removed.

1. National Criminal Investigation DNA Database (NCIDD)-Integrated Forensic Analysis (NIFA)

The NIFA is a national DNA database maintained by the Australian Criminal Intelligence Commission (ACIC) for conducting national searches of autosomal STR, Y chromosome STR and mitochondrial SNP profiles to link UHR and relatives of LTMP.

2. International Criminal Police Organisation (INTERPOL) I-Familia Database

The INTERPOL I-Familia Database is an international DNA database maintained by INTERPOL for conducting international searches of autosomal STR, Y chromosome STR and mitochondrial SNP profiles to link UHR and relatives of LTMP.

3. Other International Law Enforcement DNA Databases

National law enforcement DNA databases in other countries may also be used for conducting international searches of autosomal STR, Y chromosome STR and mitochondrial SNP profiles to link UHR and relatives of LTMP.

What will happen to genetic information obtained from unidentified human remains?

The deoxyribonucleic acid (DNA) profiles from unidentified human remains (UHR) samples will be subject to a number of genetic analyses to aid the process of identifying them. These include:

• Direct DNA searching and matching using one or more genetic targets such as autosomal short tandem repeats (STRs), Y chromosome STRs and single nucleotide polymorphisms (SNPs), mitochondrial SNPs, X chromosome STRs and SNPs, and identity informative SNPs
• Short range familial/kinship searching and matching to identify close genetic relatives using one or more genetic targets such as autosomal STRs, Y chromosome STRs and SNPs, mitochondrial SNPs, X chromosome STRs and SNPs, and kinship informative SNPs
• Medium to long range familial/kinship searching and matching to identify more distant genetic relatives using kinship informative SNPs
• Estimation of genetic ancestry using ancestry informative SNPs
• Estimation of eye and hair colour using phenotype informative SNPs

DNA profiles from UHR can be compared to equivalent DNA profiles from direct or family reference samples on a case-by-case basis or they may be submitted to external databases for direct or familial/kinship searching against a collection of known DNA profiles. They will generally be utilised in the order shown below, from least information revealed from the DNA profile submitted to most information, until the identity of the UHR can be inferred.

DNA profiles submitted to law enforcement DNA databases are not publicly available and are typically regularly searched and retained on the database until the UHR has been identified. In contrast, online DNA databases may be publicly available. DNA profiles submitted to reference or population databases are typically submitted for one-off searches, not retained on the database and cannot be used to identify an individual but rather a population of individuals with the same maternal/paternal lineage, genetic ancestry or general pigmentation (database dependent). DNA profiles submitted to genetic genealogy databases are typically regularly searched and retained on the database until the UHR has been identified.

1. National Criminal Investigation DNA Database (NCIDD)

The NCIDD is a national DNA database maintained by the Australian Criminal Intelligence Commission (ACIC) for conducting national searches of autosomal STR profiles to link UHR and long-term missing persons (LTMP).

2. NCIDD-Integrated Forensic Analysis (NIFA)

The NIFA is a national DNA database maintained by the ACIC for conducting national searches of autosomal STR, Y chromosome STR and mitochondrial SNP profiles to link UHR and LTMP, or UHR and relatives of LTMP.

3. International Criminal Police Organisation (INTERPOL) DNA Database

The INTERPOL DNA Database is an international DNA database maintained by INTERPOL for conducting international searches of autosomal STR profiles to link UHR and LTMP.

4. INTERPOL I-Familia Database

The INTERPOL I-Familia Database is an international DNA database maintained by INTERPOL for conducting international searches of autosomal STR, Y chromosome STR and mitochondrial SNP profiles to link UHR and relatives of LTMP.

5. Other International Law Enforcement DNA Databases

National law enforcement DNA databases in other countries may also be used for conducting international searches of autosomal STR, Y chromosome STR and mitochondrial SNP profiles to link UHR and LTMP, or UHR and relatives of LTMP.

6. European DNA Profiling Group Mitochondrial DNA Population Database (EMPOP)

EMPOP is an international, online database maintained by the University of Innsbruck for conducting searches of mitochondrial SNP profiles from UHR to estimate the frequency of the mitochondrial SNP profile in the general population and maternal lineage.

7. Y-Chromosome STR Haplotype Reference Database (YHRD)

YHRD is an international, online database maintained by Charité Universitätsmedizin Berlin for conducting searches of Y chromosome STR profiles from UHR to estimate the frequency of the Y chromosome STR profile in the general population and paternal lineage.

8. Forensic Research/Reference on Genetics ‐ Knowledge Base (FROG‐kb)

FROG-kb is an international, online database maintained by Yale University for conducting searches of ancestry informative SNP profiles from UHR to estimate the genetic ancestry.

9. Snipper App Suite

Snipper App Suite is an international, online database maintained by the University of Santiago de Compostela for conducting searches of ancestry informative SNP profiles from UHR to estimate the genetic ancestry.

10. HIrisPlex

HIrisPlex is an international, online database maintained by Erasmus Medical Centre Rotterdam for conducting searches of phenotype informative SNP profiles from UHR to estimate the eye and hair colour.

11. Genetic Genealogy Databases

In the absence of direct and/or family reference samples, it may be necessary to find (close or distant) genetic relatives of UHR using forensic investigative genetic genealogy. This technique involves conducting international searches of autosomal SNP profiles from UHR using one or more international, online, law enforcement accessible genetic genealogy databases.

The two genetic genealogy databases commonly used by the AFP and Missing Persons Units are:

• GEDmatch PRO – this is a dedicated law enforcement portal maintained by Verogen (QIAGEN), a forensic genomics company based in California, USA. The portal is designed to support law enforcement with investigative comparisons to DNA profiles on GEDmatch, a public genetic genealogy database also maintained by Verogen, and separate these activities from standard genealogy research activities. DNA profiles available for searching and matching by the National DNA Program for Unidentified and Missing Persons through GEDmatch PRO include those from all individuals who have submitted their DNA profiles to GEDmatch from other consumer genomics service providers (e.g. AncestryDNA, 23andMe, MyHeritage, Living DNA, FamilyTreeDNA). This is because all GEDmatch DNA profiles can be used by any law enforcement agency across the world for identifying UHR. UHR DNA profiles submitted to GEDmatch PRO will not appear in match lists generated for any public user.
• FamilyTreeDNA – this is a public genetic genealogy database maintained by Gene by Gene and Othram, consumer/forensic genomics companies based in Texas, USA. DNA profiles available for searching and matching by the National DNA Program for Unidentified and Missing Persons include those from individuals who have submitted their DNA to FamilyTreeDNA for consumer genomics testing or have submitted their DNA profiles to FamilyTreeDNA from other consumer genomics service providers (listed above) and who have given permission for their DNA profiles to be used by any law enforcement agency across the world. UHR DNA profiles submitted to FamilyTreeDNA will not appear in match lists generated for any public user.

The more deoxyribonucleic acid (DNA) that is tested by law enforcement agencies and/or consumer/forensic genomics service providers, the greater the possibility that information not related to the purpose of identifying unidentified human remains (UHR) is potentially available. This may include personal, sensitive or health related information, such as:

• Medical conditions and risks
• Unknown or unexpected genetic relatives e.g. children, parents or siblings
• Other physical traits

This type of information is not be specifically targeted or analysed by the AFP, and any incidental information that is derived from DNA profiles produced will not be disclosed to long-term missing persons (LTMP) (if located alive) or their genetic relatives.

Previously unknown, unexpected or unwanted personal, sensitive or health related information may become available to relatives of LTMP or members of the public that undertake consumer genomics testing and voluntarily elect to upload their consumer genomics DNA profile to genetic genealogy databases. For example, they may discover that a family member is not a genetic relative, is deceased, has been the victim of a crime or has a criminal history.

Consumer/forensic genomics service providers and private/public genetic genealogy databases have their own terms of service/use and privacy policies related to the collection, use and disclosure of genetic and other personal information. Individuals should read and understand the relevant terms and policies before participating in these services and submitting genetic information to these databases.

Direct and family reference samples, and any deoxyribonucleic acid (DNA) profiles derived from these samples, are stored securely by the Australian Federal Police (AFP). Only authorised AFP personnel will have access to the secure laboratories, offices and password-protected computers that house these samples and the resulting DNA profiles.

The Australian Federal Police (AFP) has implemented a number of steps to manage, minimise or eliminate any privacy impacts to individuals. Some of these privacy enhancing steps are detailed below.

Direct reference samples, family reference samples and unidentified human remains (UHR) samples are assigned a unique Forensic Exhibit Number (FEN) upon receipt at the AFP laboratory. This unique FEN, as well as unique FENs assigned to any sub-samples generated during the examination, are used throughout the deoxyribonucleic acid (DNA) testing process. This ensures the DNA profiles, presented in the form of an alphanumerical code, are de-identified through the use of a unique FEN prior to them being submitted to any national, international or genetic genealogy databases as appropriate. Furthermore, upon submission of UHR DNA profiles to genetic genealogy databases, the DNA data files are reconfigured and/or encrypted and unable to be accessed by other users.

The AFP use a range of DNA techniques for the purpose of identifying UHR. The general principle followed is that of sequential unmasking of genetic information. The AFP will initially use routine DNA technologies (such as autosomal short tandem repeat (STR) and Y chromosome STR profiling), which target relatively small areas of the human genome and/or reveal relatively little information about the UHR, long-term missing person (LTMP) or LTMP relative(s). If those technologies are sufficient to make an identification, the DNA testing process will stop.

If, however, they are insufficient or deemed unsuitable due to inadequate DNA quality or quantity, the DNA testing process will continue and the AFP may use specialist DNA technologies (such as mitochondrial single nucleotide polymorphism (SNP) profiling, autosomal SNP profiling, ancestry and phenotype prediction and/or forensic investigative genetic genealogy), which target a larger part of the genome and/or potentially reveal more information about the UHR, LTMP or LTMP relative(s).

The AFP has service contracts and confidentiality agreements in place with all external service providers and consultants. This includes contracted forensic genomics service providers who perform the whole genome sequencing and contracted genetic genealogists who perform the genealogy research for the forensic investigative genetic genealogy process. The service contracts and confidentiality agreements place parameters around how the genetic and other personal information can be used and disclosed.
 

Genetic information derived from deoxyribonucleic acid (DNA) profiles produced will not be disclosed to long-term missing persons (LTMP) (if located alive) or their genetic relatives. More information about the Australian Federal Police’s (AFP’s) handling of personal information (including how to seek access to records or make a complaint) can be found in the AFP’s Privacy Policy or by contacting the AFP’s Privacy Officer (Privacy@afp.gov.au).

An individual can withdraw consent to the handling of their deoxyribonucleic acid (DNA) or genetic information by the AFP at any time by contacting their Case Officer, or the Missing Persons Unit in their state or territory, if they collected the reference DNA sample. An individual can withdraw consent to law enforcement searching and matching and/or remove their DNA data from genetic genealogy databases by adjusting their database account settings accordingly.