Difference between revisions of "Team:Bordeaux/Software"

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<p>

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These fastq files are the input for the HISAT software, based on bowtie, it performs the mapping of the reads on the genome. HISAT was used with the parameters ~~previously described in the work of Denis Dupuy that produced the reference junctions file (ref)~~. HISAT outputs bam files, they are a binary version of a sam file which contains the mapping informations like localisation of sequences reads sequences.

+

These fastq files are the input for the HISAT software, based on bowtie, it performs the mapping of the reads on the genome. HISAT was used with the default parameters. HISAT outputs bam files, they are a binary version of a sam file which contains the mapping informations like localisation of sequences reads sequences.

</p>

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Since the biology team had not produced any results of RNA-Seq, we had to choose a training dataset from Mae et al, which is composed of stages and muscle specific RNA-Seq reads. A very useful asset in order to detect tissue specific splicing patterns.</p>

−

<p>If the biology team had produced a modified <i>C.elegans</i> worm, we would have been interested in checking if other gene splicing were impacted by the genetic construct. We therefore compared muscle and neuron alternative splicing patterns in order to identify specific genes which could be responsible for the differentiation in one of the tissue studied.

+

<p>If the biology team had produced a modified <i>C.elegans</i> worm, we would have been interested in checking if other gene splicing were impacted by the genetic construct and verify if unc-60 splicing was modified. We therefore compared muscle and neuron alternative splicing patterns in order to identify specific genes which could be responsible for the differentiation in one of the tissue studied.

−

~~It could also have been possible to compare RNA-Seq samples from our worms to neuron or muscle specific WT patterns and detect modified junction usages~~.

+

</p>

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<h3>3.1. ~~Validating the efficiency~~ of ~~the~~ pipeline results</h2>

+

<h3>3.1. Evaluation of pipeline results</h2>

<p>First of all, to confirm the efficiency of our workflow we decided to look for housekeeping genes behaviors. Among all these genes we have chosen the actin-3. As expected we have been able to locate its junctions in the diagonal area meaning that this particular gene does not have a different alternative splicing between the neuron and muscle. Thus we confirmed the robustness of our pipeline and that allowed us to perform more analysis which are discussed in the following lines.</p>

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<h3>3.2. unc-60 splicing investigation</h2>

−

<p>Since we knew a priori the behavior of unc60, it was an interesting positive control to investigate. We can see on the plot that muscular isoform B and non-muscular isoform A usages behave as expected. Indeed, in the muscle, the usage ratio for ~~UNC~~-60B is 0.98 versus 0.02 for ~~UNC~~-60A, a very dichotomic junction usage reflecting the muscle isoform specificity. In contrast, the ~~usages~~ ratios for both isoforms are neighbouring 0.5, which would indicate that both isoforms are used ~~in neuron~~.</p>

+

<p>Since we knew a priori the behavior of unc60, it was an interesting positive control to investigate. We can see on the plot that muscular isoform B and non-muscular isoform A usages behave as expected. Indeed, in the muscle, the usage ratio for unc-60B is 0.98 versus 0.02 for unc-60A, a very dichotomic junction usage reflecting the muscle isoform specificity. In contrast, the usage ratios for both isoforms in neuron are neighbouring 0.5, which would indicate that both isoforms are used.</p>

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<h3>3.3. ric-4 splicing investigation</h2>

−

<p>We had no a priori knowledge about ric-4 but it caught our attention since its behavior is very characteristic of an outlier. Actually its two isoforms are located on the opposite of the diagonal meaning an inversion of spliced forms in comparison with the genes located in the central area. We can see one form very used in the neuron whereas the other one is more used in the muscular tissue.We then investigate the role of ric-4~~. Thus we found that this gene is involved in the structuration of synapses and their functions~~.

+

<p>We had no a priori knowledge about ric-4 but it caught our attention since its behavior is very characteristic of an outlier. Actually its two isoforms are located on the opposite of the diagonal meaning an inversion of spliced forms in comparison with the genes located in the central area. We can see one form very used in the neuron whereas the other one is more used in the muscular tissue. We then investigate the role of ric-4.

−

~~ric-4~~ is thought to be related to vesicles trafficking including SNARE vesicles. It is tagged as involved in synapses structuration and function. However SNARE vesicles processes are also found in muscle. Therefore muscle and neuron specific isoforms of these vesicular transport related proteins could exist.</p>

+

It is thought to be related to vesicles trafficking including SNARE vesicles. It is tagged as involved in synapses structuration and function. However SNARE vesicles processes are also found in muscle. Therefore muscle and neuron specific isoforms of these vesicular transport related proteins could exist.</p>

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<h3>3.4. rsr-1 splicing investigation</h2>

−

<p>rsr-1 was picked up because it presents a splicing pattern very similar to ~~UNC~~-60. Indeed, rsr-1 isoforms in muscle have poles-apart usage ratios (0.98 vs 0.02) while in neuron this dichotomic usage is quite less pronounced (0.65 vs 0.35). rsr-1 is a homolog of SR160m, a splicing co-activator. It is important for development including normal pharyngeal morphology.

+

<p>rsr-1 was picked up because it presents a splicing pattern very similar to unc-60. Indeed, rsr-1 isoforms in muscle have poles-apart usage ratios (0.98 vs 0.02) while in neuron this dichotomic usage is quite less pronounced (0.65 vs 0.35). rsr-1 is a homolog of SR160m, a splicing co-activator. It is important for development including normal pharyngeal morphology.

−

In Ensembl database this gene is featuring only one splice variant. We obtained 7 and 229 read counts for muscular isoforms, and 7 and 13 for the neuron. The few read counts could be due to mapping errors, revealing alternative junctions that are not actually real. This is possible in regions of lower complexity. rsr-1 actually ~~present~~ a low complexity region, long serine and arginine repeats.</p>

+

In Ensembl database this gene is featuring only one splice variant. We obtained 7 and 229 read counts for muscular isoforms, and 7 and 13 for the neuron. The few read counts could be due to mapping errors, revealing alternative junctions that are not actually real. This is possible in regions of lower complexity and rsr-1 actually presents a low complexity region, long serine and arginine repeats.</p>

Difference between revisions of "Team:Bordeaux/Software"

Latest revision as of 20:40, 1 November 2017

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1. What is RNA-Seq ?

2. How to study splicing with RNA-Seq ?

2.1. Aligning & Mapping

2.2. Extracting junctions

2.3. Calculating ratios

2.4. Plotting the results

3. Analysing the results

3.1. Evaluation of pipeline results

3.2. unc-60 splicing investigation

3.3. ric-4 splicing investigation

3.4. rsr-1 splicing investigation

Discussion and future improvements

How to find us ?

Mail: igembdx@gmail.com

Copyright © iGEM Bordeaux 2017

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+.ourWorks * {width : 85%; margin: 2% auto; color: #E0E0E0}
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 .ourWorks h1 {padding-left: 20px; font-size: 30px}
@@ Line 66: / Line 66: @@
 <p>
-   These fastq files are the input for the HISAT software, based on bowtie, it performs the mapping of the reads on the genome. HISAT was used with the parameters previously described in the work of Denis Dupuy that produced the reference junctions file (ref). HISAT outputs bam files, they are a binary version of a sam file which contains the mapping informations like localisation of sequences reads sequences.
+   These fastq files are the input for the HISAT software, based on bowtie, it performs the mapping of the reads on the genome. HISAT was used with the default parameters. HISAT outputs bam files, they are a binary version of a sam file which contains the mapping informations like localisation of sequences reads sequences.
 </p>
@@ Line 110: / Line 110: @@
 Since the biology team had not produced any results of RNA-Seq, we had to choose a training dataset from Mae et al, which is composed of stages and muscle specific RNA-Seq reads. A very useful asset in order to detect tissue specific splicing patterns.</p>
-<p>If the biology team had produced a modified <i>C.elegans</i> worm, we would have been interested in checking if other gene splicing were impacted by the genetic construct. We therefore compared muscle and neuron alternative splicing patterns in order to identify specific genes which could be responsible for the differentiation in one of the tissue studied.
+<p>If the biology team had produced a modified <i>C.elegans</i> worm, we would have been interested in checking if other gene splicing were impacted by the genetic construct and verify if unc-60 splicing was modified. We therefore compared muscle and neuron alternative splicing patterns in order to identify specific genes which could be responsible for the differentiation in one of the tissue studied.
-It could also have been possible to compare RNA-Seq samples from our worms to neuron or muscle specific WT patterns and detect modified junction usages.
 </p>
@@ Line 123: / Line 122: @@
 <img style="width:500px; margin-left:auto; margin-right:auto; display:block" src="https://static.igem.org/mediawiki/2017/thumb/0/03/Bdx-all.png/655px-Bdx-all.png">
-<h3>3.1. Validating the efficiency of the pipeline results</h2>
+<h3>3.1. Evaluation of pipeline results</h2>
 <p>First of all, to confirm the efficiency of our workflow we decided to look for housekeeping genes behaviors. Among all these genes we have chosen the actin-3. As expected we have been able to locate its junctions in the diagonal area meaning that this particular gene does not have a different alternative splicing between the neuron and muscle. Thus we confirmed the robustness of our pipeline and that allowed us to perform more analysis which are discussed in the following lines.</p>
@@ Line 129: / Line 128: @@
 <h3>3.2. unc-60 splicing investigation</h2>
-<p>Since we knew a priori the behavior of unc60, it was an interesting positive control to investigate. We can see on the plot that muscular isoform B and non-muscular isoform A usages behave as expected. Indeed, in the muscle, the usage ratio for UNC-60B is 0.98 versus 0.02 for UNC-60A, a very dichotomic junction usage reflecting the muscle isoform specificity. In contrast, the usages ratios for both isoforms are neighbouring 0.5, which would indicate that both isoforms are used in neuron.</p>
+<p>Since we knew a priori the behavior of unc60, it was an interesting positive control to investigate. We can see on the plot that muscular isoform B and non-muscular isoform A usages behave as expected. Indeed, in the muscle, the usage ratio for unc-60B is 0.98 versus 0.02 for unc-60A, a very dichotomic junction usage reflecting the muscle isoform specificity. In contrast, the usage ratios for both isoforms in neuron are neighbouring 0.5, which would indicate that both isoforms are used.</p>
 <img style="width:500px; margin-left:auto; margin-right:auto; display:block" src="https://static.igem.org/mediawiki/2017/thumb/5/5b/Bdx-unc-60.png/612px-Bdx-unc-60.png">
@@ Line 135: / Line 134: @@
 <h3>3.3. ric-4 splicing investigation</h2>
-<p>We had no a priori knowledge about ric-4 but it caught our attention since its behavior is very characteristic of an outlier. Actually its two isoforms are located on the opposite of the diagonal meaning an inversion of spliced forms in comparison with the genes located in the central area. We can see one form very used in the neuron whereas the other one is more used in the muscular tissue.We then investigate the role of ric-4. Thus we found that this gene is involved in the structuration of synapses and their functions.
+<p>We had no a priori knowledge about ric-4 but it caught our attention since its behavior is very characteristic of an outlier. Actually its two isoforms are located on the opposite of the diagonal meaning an inversion of spliced forms in comparison with the genes located in the central area. We can see one form very used in the neuron whereas the other one is more used in the muscular tissue. We then investigate the role of ric-4.
-ric-4 is thought to be related to vesicles trafficking including SNARE vesicles. It is tagged as involved in synapses structuration and function. However SNARE vesicles processes are also found in muscle. Therefore muscle and neuron specific isoforms of these vesicular transport related proteins could exist.</p>
+It is thought to be related to vesicles trafficking including SNARE vesicles. It is tagged as involved in synapses structuration and function. However SNARE vesicles processes are also found in muscle. Therefore muscle and neuron specific isoforms of these vesicular transport related proteins could exist.</p>
 <img style="width:500px; margin-left:auto; margin-right:auto; display:block" src="https://static.igem.org/mediawiki/2017/thumb/8/89/Bdx-ric-4.png/593px-Bdx-ric-4.png">
@@ Line 142: / Line 141: @@
 <h3>3.4. rsr-1 splicing investigation</h2>
-<p>rsr-1 was picked up because it presents a splicing pattern very similar to UNC-60. Indeed, rsr-1 isoforms in muscle have poles-apart usage ratios (0.98 vs 0.02) while in neuron this dichotomic usage is quite less pronounced (0.65 vs 0.35). rsr-1 is a homolog of SR160m, a splicing co-activator. It is important for development including normal pharyngeal morphology.
+<p>rsr-1 was picked up because it presents a splicing pattern very similar to unc-60. Indeed, rsr-1 isoforms in muscle have poles-apart usage ratios (0.98 vs 0.02) while in neuron this dichotomic usage is quite less pronounced (0.65 vs 0.35). rsr-1 is a homolog of SR160m, a splicing co-activator. It is important for development including normal pharyngeal morphology.
-In Ensembl database this gene is featuring only one splice variant. We obtained 7 and 229 read counts for muscular isoforms, and 7 and 13 for the neuron. The few read counts could be due to mapping errors, revealing alternative junctions that are not actually real. This is possible in regions of lower complexity. rsr-1 actually present a low complexity region, long serine and arginine repeats.</p>
+In Ensembl database this gene is featuring only one splice variant. We obtained 7 and 229 read counts for muscular isoforms, and 7 and 13 for the neuron. The few read counts could be due to mapping errors, revealing alternative junctions that are not actually real. This is possible in regions of lower complexity and rsr-1 actually presents a low complexity region, long serine and arginine repeats.</p>