Download - 310 Data Collection Software
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310 Data Collection Software
Controls 310 run conditions
Translates light on CCD camera intoelectropherogram (raw data)
Sample sheets and injection lists are created
Macintosh
1.0.2
1.2.2
2.1 (5-dye)
Windows NT
Just being
released
Just being
released
ABI manual is P/N 904958B
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Injection ist in Data Collection
Software ists samples to !e anal"#ed (repeats can !e easil"
performed)
Sets $irtual filter on CCD camera
Sets electrophoresis time and $oltage
Sets injection time and $oltage
Sets run temperature
If desired% sample anal"sis can !e set up forautomatic matri& color separation and si#ing with
internal standards using defined anal"sis
parameters
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Steps 'erformed in Standard
odule Capillary fill pol"mer solution is forced into the capillar" !" appl"ing a force to
the s"ringe
Pre-electrophoresis the separation $oltage is raised to 10%000 $olts and run for *minutes+
Water wash of capillary capillar" is dipped se$eral times in deioni#ed water toremo$e !uffer salts that would interfere with the injection process
Sample injection the autosampler mo$es to position ,1 (or the ne&t sample inthe sample set) and is mo$ed up onto the capillar" to perform the injection+ a$oltage is applied to the sample and a few nanoliters of sample are pulled onto theend of the capillar"+ the default injection is 1* -. (-ilo$olts) for * seconds
Water wash of capillary capillar" is dipped se$eral times in waste water toremo$e an" contaminating solution adhering to the outside of the capillar"
Water dip capillar" is dipped in clean water (position /) se$eral times Electrophoresis autosampler mo$es to inlet !uffer $ial (position 1) andseparation $oltage is applied across the capillar"+ the injected D, molecules
!egin separating through the ''2 pol"mer solution
Detection data collection !egins+ raw data is collected with no spectraldecon$olution of the different d"e colors+ the matri& is applied during 4enescananal"sis
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(prior to separation of fluorescent dye colors)
5aw Data from the ,6I 'rism 310
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4eneScan7Software
Calls pea-s (!ased on threshold $alues) Separates colors with matri& file
Si#es pea-s with internal si#e standard
Macintosh
2.1
3.1
3.1.2 (5-dye)
Windows NT
3.7 (5-dye)
ABI manual is P/N 4303189
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Screens in 4eneScan7'rogram
'rocessed data
Si#ing data
8lectrophoresis histor" Sample Information
5aw data
,nal"sis log file
Each screen can be used to aid in evaluation of
samples and trouble shooting problem samples
during data analysis
Each screen can be used to aid in evaluation of
samples and trouble shooting problem samples
during data analysis
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atri& Standards (5aw Data)
6FAM
TET
HEX
ROX
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Sa$e & atri& Created
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DNA fragent
pea!s in saple
DNA
Size
Data
Point
"#$%&' p"#$%&' p
"6%* p"6%* p
100
13
1!01"0
#00
#!0
DNA fragment pea$s aresized based on the sizing
curve produced from the
points on the internal size
standard
Process of Siing D!" #ragments
$sing an %nternal Standard
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Si#ing ,lgorithm
ocal Southern is commonl" used !ut ma" not !e the!est in all situations
ocal Southern in$ol$es using / pea- a!o$e and /pea-s !elow an un-nown pea- from the internal si#estandard to ma-e a calculated D, si#e
+i,e of
+TR Allele
X" X' X" - X'
/
%
&nternal size standard
S'( Allele
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Internal Si#ing Standards)S!00 (*+ ,Applied -iosystems.
/'& !0!00 (*+ ,/ife 'echnologies.
&/S"00 +( ,Promega.
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Thoughts on Si#e Standards
6e consistent in use if "ou want to !e a!le tocompare data o$er time
,ll si#e standards I ha$e tested wor-
,llele si#es are different with different internalsi#ing standards
4S*00 has a large 9hole: in its si#ing a!ilit" when
using the local Southern algorithm for medium2
si#ed ST5 alleles !ecause of the /*0 !p pea- thatcannot !e used+ also must !e run out to *0 !p to
!e a!le to t"pe large ;4, alleles with ,6I -its
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4enot"per Software
Con$erts 4eneScan si#ed pea-s into genot"pe
calls using macros 4enot"ping performed !" comparison of allele
si#es in allelic ladder to sample alleles
Macintosh
2.0
2.5
2.5.2 (5-dye)
Windows NT
3.7 (5-dye)
ABI manual is P/N 904648
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.rofiler .lus Allelic /adders
D3S13!2 )A45A
A6E/ D2S117 D#1S11 D12S!1
D!S212 D13S317D7S2#0
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0Ofiler Allelic /adders
D3S13!2
A6E/
D7S2#0
D1"S!3
'801'P*+ S1P*
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2dentifiler Allelic /adders
D3S13!2 D1"S!3
45A
A6E/
D2S117D#1S11
D12S!1
D1S933
D!S212
)A
D#S1332
'P*+
'801 D13S317
S1P*D7S2#0
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.o3er.le45"6 /adders
D3S13!2
D1"S!3
45A D2S117
D#1S11 D12S!1
)A
Penta D
'P*+
D13S317S1P*
D7S2#0
A6E/
Penta E'801
D!S212
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Data 0ollection
.ea! 2dentification
Data Reie3 y
Analyst7E4ainer
0olor +eparation
.ea! +i,ing
0oparison to Allelic
/adder
0onfiration of
Results y +econd
Analyst7E4ainer
1enotype
Assignent to Alleles
)eneScan
soft:are
)enotyper
soft:are
&nternal sizing
standard
,e;g;< )S!00(*+.
6atri= file
Allelic ladder
sample
Steps in ST5 4enot"ping 'rocessData ollection
soft:are
Expert Systems
under Development
(e.g., True Allele)
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Three 'ossi!le utcomes
&atch 'ea-s !etween the compared ST5 profiles ha$e the samegenot"pes and no une&plaina!le differences e&ist !etween thesamples< Statistical e$aluation of the significance of the match isusuall" reported with the match report
N*' directly e?uivalent to 310
Subtle differences in matri=
formation and sizing algorithms >
N*' directly e?uivalent to 310
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,6I 3100 ,rra" Detection
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2dentifiler Data fro
a 8ad 0apillary in
&"** Array
2dentifiler Data fro
a 8ad 0apillary in
&"** Array
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2dentifiler Data fro
a 1ood 0apillary in
&"** Array
2dentifiler Data fro
a 1ood 0apillary in
&"** Array
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6iological 9,rtifacts: of ST5
ar-ers Stutter 'roducts
on2template nucleotide addition
icro$ariants
ull alleles
utations
Chapter 6 covers
these topics in detail
Chapter 6 covers
these topics in detail
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Stutter 'roducts
'ea-s that show up primaril" one repeat less than thetrue allele as a result of strand slippage during D,s"nthesis
Stutter is less pronounced with larger repeat unit si#es(dinucleotides = tri2 = tetra2 = penta2)
onger repeat regions generate more stutter
8ach successi$e stutter product is less intense
(allele = repeat21 = repeat2/) Stutter pea-s ma-e mi&ture anal"sis more difficult
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ST5 ,lleles with Stutter 'roducts
D#1S11 D12S!1
D2S117
DNA +i,e (p)
Stutter
Product
";3@ ";#@!;9@
Allele
Relati6eFluores
cence9nits
S h i f S ' d
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Schematic of Stutter 'roduct
;ormation 'rocess
:als; et al("
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on2template ,ddition Ta> pol"merase will often add an e&tra nucleotide
to the end of a 'C5 product+ most often an 9,:
Dependent on *?2end of the re$erse primer
Can !e enhanced with e&tension soa- at the endof the 'C5 c"cle (e
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A A
A
AAA
!BAA)C
!BAAA)C
Last Base for Primer
Opposite Dye Label
Impact of the *? nucleotide
on on2Template ,ddition
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ull ,lleles
,llele is present in the D, sample !ut fails to !e
amplified due to a nucleotide change in a primer
!inding site
,llele dropout is a pro!lem !ecause a hetero#"goussample appears falsel" as a homo#"gote
Two 'C5 primer sets can "ield different results on
samples originating from the same source
This phenomenon impacts D, data!ases
arge concordance studies are t"picall" performed
prior to use of new ST5 -its
f " S i i i
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E
E>
>
6
6 >
Allele 6 aplicon
;as @dropped out
2alance in allele
pea! ;eig;ts
Hetero,ygous alleles
are 3ell alanced
%mpact of D!" Se)uence *ariation in
the PC+ Primer ,inding Site
No mutation
6utation at 3Bend of
primer binding site
,allele dropout.
6utation inmiddle of primer
binding site
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-ocus .its Compared +esults +eference
D13 ''1
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icro$ariants Defined as alleles that are not e&act multiples of
the !asic repeat motif or se>uence $ariants of the
repeat motif or !oth
a" e&ist as insertion% deletion% or !ase change Se>uence $ariation can occur within repeat% in the
flan-ing region% or in a primer !inding site
Can cause 'C5 failure due to pol"morphism in theprimer site 22 9null alleles:
D i f i i
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'>%"'>%"
Detection of a icro$ariant
,llele at the ST5 locus ;4,
1L S/*2/*L /
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Caution with Si#ing 8&treme 9ff2adder:
,lleles
)A ladder allele 30 n$no:n )A allele bp repeat
#"9;7 330;"0 "!;21 1";9! ,9";#.
#"9;" 330;"3 "!;"7 1";9# ,9";#.
#"9;"" 330;!0 "!;29 1";9" ,9";#.
#"7;22 3#;99 "1;7" 1!;99 ,9!;#.
#"7;13 3#;2 "1;7" 1!;99 ,9!;#.
#"7;!" 3#;#3 "1;"7 1!;91 ,9!;#.
310 Data310 Data
377 Data377 Data
Data courtesy of Melissa Fieel!orn (Maine +tate .olice 0rie /a)
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Three2'ea- 'atterns
D#1S11
F'ype 1G F'ype #G
-alanced pea$heights
Sum of heights of
t:o of the pea$s is
e?ual to the third
D12S!1
Most common in
TPOX and D21S11
Most common in
D18S51 and ..
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utation !ser$ed in ;amil"
Trio
"#B">
"B">
"B"$ "#B">
"&B"$
"B"$
Noral Transission of Alleles
(No Mutation)
.aternal Mutation
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easured utation 5ates+TR /ocus Maternal Meioses (C) .aternal Meioses (C) Null Alleles (C) Multi?8anded (C)
S1P* "#7#$>#& (*%*&) &""7'#&"'# (*%"&) '7#'*'* (*%*") None reported
)A $7>'& (*%*") 7">$66 (*%*") "*7#&$# (*%*') ""7#&$*# (*%*&) "&7#'*'* (*%*&)
45A '*7>>&< (*%*&) >"7'*"&" (*%) $7#'''* (*%*') "76>" (*%*')
D3S13!2 *7#>>< (*%*') *'* (*%") "7#'*'* (*%*") "7#*6 (*%')
D2S117 766$' (*%*$) '>'$ (*%*
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Summar" of ST5 utations
utations happen and need to !e considered
Rsuall" 1 in 1000 meioses
'aternal normall" higher than maternal
.,% ;4,% and D1HS*1 ha$e highest le$els
TQ01% T'K% and D1AS*3F ha$e lowest
le$els
STRBase
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4eneral Information
Intro to ST5s(downloada!le 'ower'oint)
ST5 ;act Sheets
Se>uence Information
ultiple& ST5 Jits
.ariant ,llele 5eports
;orensic Interest Data
;6I CDIS Core oci
D,6 Standards
IST S5 /3F1
'u!lished 'C5 'rimers
2Chromosome ST5s
'opulation Data
.alidation Studies
Supplemental Info
5eference ist
Technolog" 5e$iew
,ddresses for Scientists
in-s to ther e! Sites
httpH:::;cstl;nist;govbiotechstrbase
/718
Short 'andem (epeat DNA
&nternet Database
STRBase
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D1
A6E/
D3
D2 45A
'801
D#1
)A D1" D12 D#
D1A6E/ D3
D2 45A
'801
D#1
)A
D1"
D12 D#
1! years old ,room temp storage.
" years old ,#0 o storage.
Results with SGM lus STR !it (Applied "iosystems)
FDecay curveG of
degraded DNA
Degraded D, 5esults
S l &i t E l
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D!S212 D13S317
D7S2#0
D2S117 D#1S11 D12S!1
Amel
45A)AD3S13!2 lue panel
!reen panel
"ello# panel
(elative3luorescenc
Sample &i'ture E'ample'rofiler 'lus data
8igher than
e=pected stutter
8igher than
e=pected stutter
FStutterG on :rong
side of allele
FStutterG on :rong
side of allele&mbalance in +
and I pea$ ratios
&mbalance in +
and I pea$ ratios
9 pea$s at a
single locus
9 pea$s at a
single locus