310 Data Collection Software

8/9/2019 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


a 8ad 0apillary in

&"** Array


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a 1ood 0apillary in

&"** Array


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

310 Data Collection Software

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