The document discusses the processes and technologies used in DNA typing and interpretation, focusing on short tandem repeat (STR) amplification and analysis methods such as peak detection thresholds and genotyping software. It outlines the structure and functions of the Combined DNA Index System (CODIS), including its various indexes for offenders, arrestees, and forensic profiles, as well as the requirements for DNA databases. Additionally, the text highlights challenges in the interpretation of DNA results, including potential artifacts from biological and technological sources.

1. Interpretation Of DNA
Typing Results
And
CODIS
SUBMITTED BY:
NEHA AGARWAL
M.Sc. Forensic science
LNJN NICFS, DELHI

2. Short tandem repeat (STR) amplification products
labeled with fluorescent dyes are separated and
detected.
Commonly used instrument approaches for collecting
the STR data. The data collection process leaves the
analyst with only a series of peaks in a CE
electropherogram (or bands on a gel). The
peak information (DNA size and quantity) must be
converted into a common language that will allow
data to be compared between laboratories. This
common language is the STR genotype.

4. Peak detection and interpretation
thresholds
Data produced in any analytical instrument will contain baseline ‘ noise ’
like static on a radio. In the case of a radio signal, the message cannot
be clearly understood unless it is louder than the static. In forensic DNA,
the signal observed must be loud and clear and reliably reflect the DNA
molecules present in the sample being tested. Peak detection thresholds
are set on capillary electrophoresis instruments below which any data is
considered unreliable. A common peak detection threshold is 50
relative fluorescence units (RFUs). Only peaks above this user-
defined analytical threshold are considered an analytical signal
and thus recorded by the data analysis software. Current software only
permits a single threshold to be applied across the entire
electropherogram.
A second threshold, called an interpretation thres hold is used in
many forensic DNA laboratories. This interpretation threshold is
sometimes referred to as the stochastic threshold , a point above which
there is a low probability that the second allele in a truly heterozygous
sample has not dropped out. If all observed peaks at an STR locus are
above the interpretation threshold, then there is confi dence that all
amplified alleles are being detected

6. Peak sizing: From data point (time) to
DNA size (bp)
• Once peaks have been characterized as being real data because
they are above the analytical threshold, they are converted
from data points in time-space (usually minutes) to DNA
size-space (base pairs, bp) so results can be compared
between separate runs. The fluorescently labeled DNA fragments
represented by peaks in capillary electropherograms (or bands on
a gel) are sized relative to an internal size standard that is mixed
with the DNA samples prior to analysis
• The internal size standard is labeled with a different colored dye
so that it can be spectrally distinguished from the DNA fragments
of unknown size. A calibration curve is established with each
sample using the known sizes of the internal size standard. Data
points from peaks in the electropherogram are then transformed
into DNA sizes relative to the internal size standard calibration
curve. A method known as Local Southern sizing is commonly
used to perform this time-to-size transformation.

8. STR genotyping: from DNA size (bp) to
allele call (# repeats)
• Following the sizing of all peaks above the analytical
threshold, the peaks in each sample are converted from DNA
size to STR allele through the use of allelic ladders. The
ladder alleles are PCR-amplified with the same primers as
provided in the STR typing kit for testing unknown samples.
Thus, samples amplified with a kit will produce alleles that are
the same size as an allele in the allelic ladder.
• The data analysis software enables a conversion of all peaks in
samples being processed from DNA size (relative to a
common internal size standard) to repeat number. The DNA
sizes of the allelic ladder alleles are used to calibrate size
ranges for allele classification. A common size range for the
genotyping allele bins is 0.5 bp

10. Genotyping Software
• In ABI 310_sample processing was performed in two steps
by two different software programs: GeneScan and
Genotyper
• GeneScan software: 1) spectrally resolve the dye colors for
each peak 2) to label peaks above a minimum analytical
threshold, and 3) to size the DNA fragments in each
sample.
• The resulting electropherograms were then imported into a
second software program where genotyping was performed.
• Genotyper program: 1) determined each sample’s genotype
by comparing the sizes of alleles observed in a standard
allelic ladder sample to those obtained at each locus tested
in the DNA sample
• GeneMapper ID software

11. FACTORS AFFECTING GENOTYPING
RESULTS
• Issues: biology related and technology related.
• Biology-related artifact peaks:
▫ Stutter products: When STR loci are PCR-amplified, a minor product peak
four bases ( n -4) shorter than the corresponding main allele peak is
commonly observed in tetra nucleotide repeats
▫ Incomplete 3(+A) nucleotide addition results with amplifications
containing too much DNA template or thermal cycling conditions that affect
the optimization of the PCR reaction. when incomplete 3 nucleotide addition
occurs, ‘ split peaks ’will result , sometimes referred to as / A, or N and N 1
peaks. In these cases, the allele of interest will be represented by two peaks 1
bp apart.
▫ Triallelic patterns result from extra chromosomal fragments being present
in a sample or the DNA sequence where the primers anneal being duplicated
on one of the chromosomes.
▫ Mixed sample results are observed if more than one individual contributed
to the DNA profile. Mixtures are readily apparent when multiple loci are
examined. An analyst looks for higher-than-expected stutter levels, more than
two peaks at a locus of equivalent intensity, or a severe imbalance in
heterozygote peak intensities.

12. • Technology-related artifact ‘ peaks ’
▫ Matrix (multicomponent) failure is a result of the inability of the
detection instrument to properly resolve the dye colors used to label
STR amplicons. This phenomenon is due to spectral overlap. A
peak of another color is ‘ pulled up ’or ‘ bleeds through‘ as a result
of exceeding the linear range of detection for the instrument (i.e.,
sample overloading)
▫ Dye blobs occur when fluorescent dyes come off of their respective
primers and migrate independently through the capillary. Most dye
artifacts, which are created through incomplete attachment of the
fluorescent dye during oligonucleotide (primer) synthesis, are typically
removed or significantly reduced through primer purification
performed by the STR kit manufacturer
▫ Air bubbles, urea crystals, or voltage spikes can give rise to a
false peak, usually sharp and appear equally intense throughout all
four colors
▫ Sample contaminants, materials that fluoresce in the visible region
of the spectrum (500 to 600 nm), may interfere with DNA typing

14. • Three parts of the genotyping process are crucial to the success of
genotyping samples.
▫ The matrix file (sometimes termed a spectral calibration) is
critical for proper color separation in an electropherogram. If the
observed peaks are not associated with the proper dye label, then the
sample genotype cannot be correctly determined.
▫ The internal size standard is necessary for proper sizing of DNA
fragment peaks detected in an electropherogram. If any of the peaks in
the size standard are below the peak detection threshold established
in the data collection and analysis software, then the sizing algorithms
will not work properly and STR alleles may be sized incorrectly.
▫ The allelic ladder is the standard to which STR alleles are compared
to obtain the sample genotype. The alleles in an allelic ladder need to
be resolved from one another and above the peak detection threshold
of the data collection and analysis software in order to correctly call
STR alleles in unknown samples. The sizes obtained for each allele in
the allelic ladder are used to make the final genotype determination in
the unknown samples. Therefore, they must be determined correctly.

15. CODIS

16. HISTORY
• Forensic DNA databases are networks for exchanging
information among law enforcement agencies to assist in
solving crimes.
• In 1995, the United Kingdom established the world’s first
national DNA database, NDNAD, in England and
Wales. Scotland and Northern Ireland have their own
databases but also submit their profiles to NDNAD.
NDNAD demonstrated initial success in solving crimes.
• Three years later, the United States introduced its
national Combined DNA Index System or CODIS.
By the end of 1998, other countries (such as Austria,
Germany, the Netherlands, New Zealand, and Slovenia)
had also introduced national DNA databases

18. Definitions
• A database is a collection of computer fi les
containing entries of DNA profiles that can be
searched to look for potential matches.
• A databank is a collection of the actual
samples — usually in the form of a blood sample
or buccal swab or their dna extracts.
• A population database includes information
on allele frequencies from a group or groups of
representative samples is included in

19. ASPECTS OF A NATIONAL DNA DATABASE
• A commitment on the part of each state (and local)
government to provide samples for the DNA database —
both offender and crime scene samples;
• A common set of DNA markers or standard core set so
that results can be compared between all samples
entered into the database;
• Standard software and computer formats so that data
can be transferred between laboratories and a secure
computer network to connect the various sites involved
in the database (if more than one laboratory is
submitting data); and
• Quality standards so that everyone can rely on results
from each laboratory.

20. Forensic DNA databases
• three parts: (1) collecting specimens from known
criminals or other qualifying individuals as defi
ned by law, (2) analyzing those specimens and
placing their DNA profi les in a computer
database, and (3) subsequently comparing
unknown or ‘ Q ’ profi les obtained from crime
scene evidence with the known or ‘ K ’profi les in
the computer database.

21. Indexes of CODIS
• The DNA profiles entered in CODIS are
organized into categories known as indexes
• The Convicted Offender Index contains DNA
profiles of individuals convicted of crimes. The
• Arrestee Index contains DNA profiles of
arrested individuals.
• The Forensic Index contains DNA profiles,
also known as forensic profiles, derived from
crime scene evidence, potentially originating
from perpetrators but not including suspects.

22. • The FBI also established the National Missing Person DNA
Database (NMPDD) Program for the identification of missing
and unidentified persons at the national level. the NMPDD are
categorized into three indexes:
• The Missing Person Index and the Unidentified Human
Remains Index contain DNA profiles from missing persons
and unidentified human remains, respectively. The
• Biological Relatives of the Missing Person Index
contains DNA profiles voluntarily contributed from relatives.
This index may also store patrilineal or matrilineal DNA profiles
from the relative such as a biological father, mother, or child of
the missing person to assist investigations.
• Additionally, a pedigree chart (a diagram showing the
relationship between the missing person and relatives) can be
created.
Indexes of CODIS

23. Indexes of CODIS
• In 2010, the FBI established a Rapid DNA Program
Office for the purpose of developing rapid DNA
technology. Rapid DNA technology is a fully automated
process of performing STR analysis within 1–2 h to
generate a CODIS core STR profile from a reference
sample
• The Rapid DNA Index System (RDIS) is the
proposed index of NDIS. It shall be an integrated system
capable of applying rapid DNA technology and carrying
out database searches from police custody or booking
units by trained police officers. The entire process
including obtaining any match from a database search,
shall take less than 2 h.

24. CODIS software
• CJIS WAN (Criminal Justice Information Systems Wide Area
Network), a stand-alone law enforcement computer network that operates
in a similar fashion to the Internet. The software is the same at all sites with
various configurations that permit different levels of access (LDIS, SDIS, or
NDIS). Software versions are updated periodically and provided to all CODIS
laboratories.
• This software enables NDIS participating laboratories to submit DNA profiles
for the 13 CODIS core STR loci to the U.S. national DNA database.
• Public crime laboratories in the United States are connected via the FBI’s
Criminal Justice Information Services Wide Area Network (CJIS WAN)
through T1 lines capable of transmitting 1.5 megabytes of information
per second. CJIS WAN provides Internet-like connectivity but without the
security risk. This network is an intranet with access only granted to
participating laboratories.

25. DNA Profiles
• The CODIS software supports the storage and search of
DNA profiles of short tandem repeat (STR), Y chromosome
STR (Y-STR), and mitochondrial DNA (mtDNA).
• Y-STR and mtDNA profiles may only be searched within
NMPDD-related indexes.
• The CODIS software no longer supports searches of DNA
profiles generated by restriction fragment length
polymorphism (RFLP) analysis.
• The 13 core CODIS STR loci are CSF1PO, FGA, THO1,
TPOX, VWA, D3S1358, D5S818, D7S820, D8S1179, D13S317,
D16S539, D18S51, and D21S11
• 7 additional CODIS STR loci are: D1S1656 D2S441 D2S1338
D10S1248 D12S391 D19S433 D22S1045 (jan,2017)

26. Routine Database Searches for
Forensic Investigations
• Currently, DNA profiles uploaded to NDIS are
automatically searched once a week (Figure 24.5). A
hit is a match made from the information provided
by comparing a target DNA profile against the DNA
profiles contained in the database. There are two
types of CODIS hits: an offender hit provides the
identity of a potential suspect of a crime, while a
forensic hit reveals the linkage between two or
more crime scenes.
• Investigation-aided cases are those assisted by
CODIS hits, including case-to-case matches as well
as case-to-offender matches

28. Search Stringency and Partial Matches
• Database searches are carried out using the CODIS
software with three stringency levels
▫ high-stringency match
▫ moderate-stringency match
▫ low-stringency search
During forensic DNA analysis, DNA degradation may prevent
full DNA profiles from being processed, producing only
partial profiles. Additionally, mixture profiles derived from
forensic samples containing DNA contributed by more than
one individual may be encountered. Furthermore, due to
mutations, null alleles may occur in the profiles produced
with some primer sets but not other primers

29. Familial Searches
• databases may be utilized to identify perpetrators by
finding a close relative, if the close relative has been
convicted of a crime and is listed in the database.
Familial search is an intentional search of a target
crime scene profile against an offender database to
obtain a list of candidate profiles that are similar to
the target profile. This list may include the profile of
a close relative of the perpetrator, who is the source
of crime scene evidence. These matches most
frequently involve siblings, parents, or children