[miso-users] questions for MISO

[Jian Duke]

Hi Yarden,
I am looking for the defferential alternative splicing events between cell
types. I am new to MISO and want to ask some basic questions before I start
to run MISO.

1) I am going to install MISO and related softwares in my desktop. What is
the requirements for the hardware to run MISO smoothly?

2) My datasets of one cell type are paired-end, but the datasets of the
other cell type are single-end. I am going to perform the mapping with
Tophat and then use the Bam files as input to run MISO. Can I compare the
paired-end data against the single-end data with MISO? Or should I just use
the forward reads of the paired-end dataset for mapping with Tophat and
take the output Bam as single-end input? Or do you have a better way?

3) I am going to run Tophat at the web-based Galaxy system. Below is the
setting for Tophat. Tophat uses the built-in reference geneome at Galaxy.
In addition, I used Illumina iGenome version of gene annotation (way to
upload: Galaxy hmepage > Shared Data > Data Libraries > iGenomes > mm9 >
genes.gtf) during the run. Do you think this settings will conflict against
downstream steps with MISO? However, I can choose not to use the iGenome
gene annontation. If I select "Use Own Junction" as No, the "Use Gene
Annotation Model" will not appear and will set as No. Should I "Use Gene
Annotation Model" when run Tophat?


Step 2: Tophat for Illumina

Will you select a reference genome from your history or use a built-in
index?: Use a built-in index

Select a reference genome: /galaxy/data/mm9/bowtie_index/mm9

Is this library mate-paired?: Single-end

TopHat settings to use: Full parameter list

Library Type: FR Unstranded

Anchor length (at least 3): 8

Maximum number of mismatches that can appear in the anchor region of
spliced alignment: 1

The minimum intron length: 70

The maximum intron length: 500000

Allow indel search: Yes

Max insertion length: 3

Max deletion length: 3

Maximum number of alignments to be allowed: 20

Minimum intron length that may be found during split-segment (default)
search: 50

Maximum intron length that may be found during split-segment (default)
search: 500000

Number of mismatches allowed in the initial read mapping: 1

Number of mismatches allowed in each segment alignment for reads mapped
independently: 1

Minimum length of read segments: 18

Use Own Junctions: Yes

Use Gene Annotation Model: Yes

Gene Model Annotations: iGenome version of gene reference mm9

Use Raw Junctions: No

Only look for supplied junctions: No

Use Closure Search: No

Use Coverage Search: Yes

Minimum intron length that may be found during coverage search: 50

Maximum intron length that may be found during coverage search: 20000

Use Microexon Search: No



Thanks in advance,

Jian
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