Tomato finishing workshop
Download
1 / 34

Tomato Finishing Workshop - PowerPoint PPT Presentation


  • 128 Views
  • Uploaded on

Tomato Finishing Workshop. April 2008 Specific Problems and Examples. Other Strategies for Finishing Large Repeats. High Repeat Content Clones and Alternative Libraries Large Insert Libraries and TILs Reading coverage histogram in gap4 and diploid plot BLAST & Pre-analysis Scaffolds

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about ' Tomato Finishing Workshop' - peers


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
Tomato finishing workshop

Tomato Finishing Workshop

April 2008

Specific Problems and Examples


Other strategies for finishing large repeats
Other Strategies for Finishing Large Repeats

  • High Repeat Content Clones and Alternative Libraries

  • Large Insert Libraries

  • and TILs

  • Reading coverage histogram in gap4 and diploid plot

  • BLAST & Pre-analysis

  • Scaffolds

  • Size using restriction digests and dotter


Gaps and assembly problems caused by repeats

Repeat

Unit

Size of

Unit

Copy

Number

Direct or

Inverted

Copies

How

Conserved?

Lower Complexity

e.g. Di-nucleotide Runs

Higher Complexity

e.g. LTRs

Gaps and Assembly ProblemsCaused by Repeats

Varying complexity of repeats depending on:

Importance of visualising repeat sequence to assess repeat type

Use Restriction Digests that cut in-frequently in repeat

Alter phrap parameters for more stringent assembly

Alternative library sizes if necessary



Orchid functionality
Orchid Functionality

  • View Template List

  • Select templates from different plates

  • View Read Pairs

  • Show only good read pairs between or within contigs

  • Show only bad read pairs

  • select between too large, too small, wrong orientation

  • Select bad readpairs across a given reading > save to file


Unspanned gap example
Unspanned gap example

Original 4-6Kb library



Restriction fragment for sil candidate
Restriction Fragment for SIL Candidate

Corresponds to position of unspanned gap


Sequencing chemistries and additives used in finishing
Sequencing Chemistries and Additives used in Finishing

  • 4:1 mix ratio of AB Big Dye Terminator : AB dGTP Terminator

  • used for general finishing reactions, not problem specific

  • AB dGTP Terminator

  • used for di-nucleotide runs and inverted repeats

  • Additive A (SequenceRx Enhancer Solution A - Invitrogen)

  • Dimethyl sulfoxide (DMSO)

  • Additive A+DMS0+dGTP

  • used for mono-nucloetide runs, inverted repeats

  • Sequence Finishing Kit (SFK) (TempliPhi - Amersham)

  • used to increase DNA yield

  • useful for structural problems caused by inverted repeats

  • GC regions


TA Repeats

Standard BDT chemistry continues to call TA even after the di-nucleotide run has ended, as is shown in the cut-off data:

Figure.1



dGTP

BDT

4:1

dGTP


In addition to di-nucleotide runs other simple repeats can also be difficult to sequence through

dGTP

Figure.6

In simple repeats the problem is often directional, for example:

GGAGGA sequences better in the forward direction

CCTCCT sequences better in the reverse direction


SFK also be difficult to sequence through


Mononucleotide runs also be difficult to sequence through

frequently occurs with C or G runs and is often a directional issue

Fig.2


4:1 also be difficult to sequence through

dGTP

dGTP + additive A + DMSO

to resolve G/C mono runs we use dGTP + A + DMSO as standard minimum attempt

directional problem - G strand sequences easier than the C strand

reactions ordered accordingly


Inverted repeat example
Inverted Repeat Example also be difficult to sequence through

Characteristic drop in sequence visible in one direction

Opposite strand shows no drop in quality but digests suggest data missing


Sfk example small inverted repeat
SFK example – small inverted repeat also be difficult to sequence through

Dotter is used to check for any repeats at problem region

Problem visible in more than one digest

Break contig and check for read pair coverage


Sfk example small inverted repeat1
SFK example – small inverted repeat also be difficult to sequence through


Inverted repeat example resolved with different chemistries and sfk
Inverted Repeat Example also be difficult to sequence throughResolved with different chemistries and SFK

Original subclone

Original subclone + A+DMSO+dGTP

SFK with 4:1


Inverted repeat example resolved with different chemistries and sfk1
Inverted Repeat Example also be difficult to sequence throughResolved with different chemistries and SFK

Original subclone

SFK

SFK + dGTP + oligo


Sfk example small inverted repeat2
SFK example – small inverted repeat also be difficult to sequence through

Using SFK then sequencing with oligo walks from both directions resolved the repeat

Restriction Digests now confirm the assembly


Use of sils and tils
Use of SILs and TILs also be difficult to sequence through


Transposon insertion library til
Transposon Insertion Library (TIL) also be difficult to sequence through

Double Stranded

Sequencing

Vector

(pUC)

Normal sequencing from either end of insert

Read pairs ~4-6Kb apart

Inserted sequence (BAC)


Transposon insertion library til1
Transposon Insertion Library (TIL) also be difficult to sequence through

Double Stranded

Sequencing

Vector

(pUC)

Normal sequencing from either end of insert

Read pairs ~4-6Kb apart

Inserted sequence (BAC)

Transposon randomly inserts across entire plasmid

Sequence outwards from transposon insertion site


Transposon insertion library til2

TIL Read pairs overlap also be difficult to sequence through

by 9bp duplication site

Transposon Insertion Library (TIL)

Double Stranded

Sequencing

Vector

(pUC)

Sequence outwards from

transposon insertion site

Inserted sequence (BAC)

Transposon randomly inserts across entire plasmid


Inverted repeat example transposon insertion library
Inverted Repeat Example also be difficult to sequence throughTransposon Insertion Library


ad