Analysis of variations found in genomes of Escherichia coli strain K12 DH10B and strain B REL606 using SynMap and GEvo analysis: Difference between revisions

Revision as of 20:48, 12 February 2010

Background

In this exercise you will compare the genomes of two Escherichia coli strains, K12 DH10B and B REL606, using whole genome syntenic comparison and high-resolution analyses of specific genomic regions. These analyses will use CoGe's tools SynMap and GEvo respectively, and will reveal evolutionary changes between these two genomes that happened after the divergence of their lineages. While the nucleotide sequence of these genomes is identical over large expanses of their genomes, many other types of large-scale genomic change will be discovered including phage insertions, transposon transposition, and genomic insertion, deletion, inversion, and duplication events. The computational tools used to do these analyses can be used for comparing the genomes of any organisms. To learn what organisms and genomes are available in CoGe, please see GenomeView.

Generating a syntenic dotplot of two Escherichia coli strains

Synteny, in genomic terms, is defined as two or more genomic regions that are derived from a common ancestor. To identify syntenic regions, you are going generate a syntenic dotplot of the genomes of Echerichia coli strains K12 DH10B and B REL606 using SynMap. First, go to SynMap. Search for these E. coli strains in CoGe's database by typing in part of the their names in the "Name" search boxes for Organism 1 and Organism 2. For example, search for "DH10B" and "REL606" respectively, or type "escheri" in both boxes. Alternatively, use this link to load SynMap with these genomes already specified. Once SynMap has found organisms that matches these names, make sure they are selected in the Organism List. While there are several parameters that can be configured when generating a syntenic dotplot using SynMap, the default settings work well for most situations, and very well for closely related organisms. Click "Generate SynMap" to start the analysis.

A lot of processing is happening behind the scenes, but the general way a syntenic dotplot is created is:

All protein coding regions (CDS)) are extracted from each genome
These sequences are blasted against each other to identify putative homologous gene pairs
Putative homologous gene pairs are analyzed to determine if they share a collinear order between the genoems

The general principle is that the most likely and parsimonious way two genomes have a collinear series of homologous genes is those genomic regions in each organism are derived from a common ancestral genomic region (hence they are syntenic). In other words, genomic synteny is inferred by a collinear arrangement of putatively homologous genes in two or more genomic genomic regions.

Syntenic dotplot data interpretation and analysis

When finished, SynMap will display a dotplot. Each axis of the dotplot is in nucleotide units and represents one of the two genomes laid end to end. The lower-left corner represents the start of each genome (usually 'ORI' for circular bacterial genomes). Each putative homologous gene-pair is drawn as a gray dot on the dotplot with its x and y position corresponding to the genomic position of each gene in their respective genomes. Gene-pairs that have been inferred as syntenic (collinear order) are colored green. The collection of these dots appear as green line, which for the comparison of these two genomes, results in a nearly continuous green line running 45-degress up the dotplot. This means that these genomes are completely syntenic.

Syntenic dotplot of Escherichia coli strain B REL606 and strain DH10B. The genomes are laid on the axes REL606 (x-axis) and DH10B (y-axis). The numbers correspond to the individual analysis of the "breaks" in the dotplot which could be found here.

If you look closely at the syntenic dotplot of these genomes, you'll notice that the syntenic line is not perfect, and there are many "breaks" or discontinuities between them. In the figure above, several these are indicated by a numbered arrow. These breaks in the syntenic path between these genomes are due to genomic changes happening at a larger scale than a single nucleotide polymorphism, and are mostly likely due to the insertion or deletion of a many nucleotide chunk in one of these genomes. In order to accurately account and characterize these discontinuities in the dotplot, you need to perform a high-resolution analysis of these regions using GEvo.

High-resolution sequence analysis using SynMap's links to GEvo

Red cross positioned at sixth break. Clicking here will open a new page of GEvo containing sequence information of both strains at this locus. Click "Run GEvo Analysis!" to visualize syntenic genes at this location

GEvo allows you to run pairwise comparisons between multiple genomic regions where you can specify how big of a genomic region to analyze. For more information on how to use GEvo please see its help page. To analyse each of these "breaks" in the sytnenic dotplot, first click on the dotplot. This will open a new window with a close-up of the dotplot. While this is mostly a redundant function when comparing bacterial genomes, this features is important when dealing with genomes with multiple chromosomes. When this window appears use the cross-hair locator that appears when you mouse over the dotplot and place it on the green spot right before a "break". The locator will turn "red" when it is over a gene-pair that can be used as a link to GEvo. When the locator has turned red, click. For example, in order to visualize GEvo analysis of "break" number six, position the locator where the number six arrow points on dotplot. Click when the locator turns "red" or use this address: [1] to regenerate this particular analysis.

Running GEvo

After positioning the locator and clicking , a new page for GEvo will appear displaying the sequence information corresponding to our region of interest in the dotplot. These sequences are "anchor" points into these two genomes for specifying the genomic regions to be compared. When linking to GEvo, SynMap automatically sets GEvo to specify using 50,000 nucleotides to the left and right of the anchor point. By default, GEvo will use BlastZ for its sequence comparison algorithm, which is a good choice for identifying large blocks of similar sequence. These settings (~100kb of each genome; BlastZ) usually work well for an initial analysis, and all you need to do is click "Run GEvo Analysis!".

GEvo's results

Once the GEvo analysis results appear, we can begin to look for and characterize the differences between these two genomes at this syntenic region. GEvo's results will show two panels, one for each genomic region. The dashed line in the middle of each panel separates the top and bottom strands of DNA. Gene models are drawn as green arrows above and below this line, and clicking on a gene will cause its annotation to appear in a box.

The pink blocks in these panels are genomic regions identified by BlastZ as being similar in sequence composition. If you click on a pink block, a transparent wedge is drawn connecting it to its partner region in the other genome, and information about the blast hit (also known as an HSP) is shown in an information box. You can click on all the pink blocks individually to connect every region of sequence similarity, or hold the "shift" key and then click on one of the pink blocks to connect them all at once. Since we are analyzing "breaks" in our dotplot that are the result of an insertion or deletion, we expect to see at least one genomic region that is present in one genome and not the other. These indels will be evident by the pattern of transparent wedges and pink blocks.

==Zooming in on a region in GEvo

As mentioned above, when you click on the dotplot, GEvo will display 50000 nucleotides towards the left and right from the anchor position. However, this may be display too much sequence. To zoom in on a region, use side bars to restrict the region of genome being displayed. This will magnify the region of interest and allows to view minute details. To get to this analysis directly, use this link: http://tinyurl.com/yemhyzg.

High-resolution GEvo analysis of an insertion: finding direct sequence repeats

Visualizing direct repeats around a putative insertion in the genome of Escherichia coli. Results can be regenerated at: http://tinyurl.com/yemhyzg

Insertions in bacterial genome could be from exogenous DNA such as plasmids and phages or it could be the result of transposition or translocation. One way to gain evidence for a recent insertion is to look for direct repeated sequences boarding a putative inserions. These direct repeats are created at the site of insertion. Notice that on the edges of "inserted" genes in REL606, the pink wedges overlap at a common syntenic region in DH10B. This is visualized by the ends of the pink blocks overlapping slightly. These are direct repeats and evidence that an insertion happened in the genome of REL606.

To determine what the genes are in the inserion,just click on the gene models and their annotations will appear in a dialog box in GEvo. While many of the genes are annotated "hypothetical", several are annotated as phage genes. This is likely a prophage.

Differences at an insertion site

Let us consider the ninth break in the dotplot. Run GEvo Analysis for this break.

Magnify the region from 50k to 100k using side bars. Notice that a number of flagella genes are present in DH10B but are missing in REL606. The presence of direct repeats is not evident yet and we cannot conclude that insertion happened in DH10B. We will BLAST DH10B against itself to see any direct repeats. If this is indeed an insertion, then we expect to see color coded bars bordering the DNA region containing these flagella genes in DH10B. Click on "Add sequence" and copy and paste the gene ID of DH10B into "name" box in the third sequence. This option allows us to view multiple sequences at the same time. Adjust the number of sequences on left and right to view syntenic regions on all three sequences.

The sequence number could be manually adjusted by adding/deleting nucleotides from left and right. This GEvo analysis page corresponds to the analysis of ninth break and we want to see direct repeats in DH10B by BLASTing it against itself.

Click "Run GEvo Analysis!".You will see various small color coded blocks beneath the inserted genes in DH10B. Click on the blocks bordering the inserted segment. The individual blocks will interconnect and create a criss cross pattern of transparent wedges. Notice that the information box will show 100% identity between the two. This means that the repeats bordering the inserted regions are exactly the same (100% identical)

Example of comparing multiple sequences simultaneously to look for direct repeats. Notice the criss cross pattern that the wedges create. Direct repeats provide evidence that insertion happened in DH10B

These are direct repeats and evidence for insertion in DH10B. Direct repeats allows us to infer that there was a staggered cut in the DNA of DH10B followed by insertion of linear piece of DNA. The gaps at the ends were repaired and ligated. These repaired gaps became direct repeats that flank the DNA segment containing flagellar genes. Notice that there is not much support that this is a translocation. You can click on several green dots that align with ninth break on our dotplot. These regions are mostly repeats which are syntenic all over the genome and are therefore "noise" in our data.

Next, we will look at phage insertion. Consider the second break in the dotplot. Run GEVo analysis on this break. Before checking for direct repeats, determine the identity of genes inserted in DH10B. These are phage-specific genes. CP4-6 prophage has integrated its DNA at this locus as seen by CP4-6 specific integrase, DNA binding protein etc.

In DH10B, the inserted genes are CP4-6 prophage specific as seen by gene annotations.

Next we will look at an example of inversion. Consider the twelfth break on the dotplot. Run GEvo Analysis on this break. Notice the syntenic regions between 10K and 30K on DH10B. Click on the pink blocks and notice the pattern of transparent wedges that connect the syntenic regions. These genes are inverted. Notice IS10R and IS10L bordering the inverted region. These IS elements have created inverted terminal repeats (ITR) which tend to invert or flip the genes within them.

In DH10B, the insertion of IS10R and IS10L has created inverted terminal repeats i.e the IS10 (IS10R and IS10L) transposons are integrated in opposite orientations. A cross over between these two transposons has inverted the DNA segment(three genes) within it. Notice the patterns of wedges connecting the syntenic region (they cross each other). Use side bars to better visualize this inversion event

Detailed analysis of each syntenic discontinuity

Variation type	Difference in strain B REL606	Difference in strain K-12 DH10B	Evidence	Notes	Link leading to GEvo
1. Deletion	none	Deletion of ~18 genes including DNA pol II, genes in metabolic pathway, thiamine ABC transporter	pseudogenes in DH10B at deletion site.	Possible additional insertion in DH10B as evidenced by pseudogenes of yabP, RNA pol associated helicase and FruR, that are not present in REl606	tinyurl.com/yexrzpb
2. Insertion	Insertion of IS1 transposon	Insertion sequences and Prophage CP46 DNA insertion	Prophage specific genes found in DH10B	Prophage DNA insertion and IS insertions has created pseudogenes in K-12 DH10B	tinyurl.com/yd2quy7
3. Translocation in REL606 and insertion in DH10B	Insertion of IS1 sequence. Translocation of ~15 genes including lac operon and other metabolic enzymes genes	Insertion of IS3 and IS2 sequences	Translocation in REL606 as evidenced by direct repeats.Dotplot shows that the missing genes are present in DH10B but not in this locus. The syntenic region is therefore not colinear.	Pseudogenes of yaiT and yaiX were created in DH10B by transposon insertions. Insertion by translocation in REL606 was confirmed as lac operon and other metabolic genes were found in DH10B by analyzing the translocated genes on the dotplot	http://tinyurl.com/yldc83u
4. Insertion in REL606 and DNA duplication event in DH10B.	Prophage DNA and transposase insertion	Recent DNA duplication event	100% identity between paralogs in DH10B and ~98% identity between syntenic region of DH10B and REL606	Possible phage DNA insertion in REL606 as "hypothetical protein" genes were found near putative prophage tail component gene in REL606.	tinyurl.com/yea8bu6
5. Insertion	Bacteriophage DNA insertion	IS2 sequence insertion	Pseudogenes at IS2 insertion site in DH10B. Phage specific genes were found in REL606	Possible phage DNA insertion in REL606 as "Hypothetical proteins" were found near phage specific genes	tinyurl.com/yevlb2w
6. Insertion	Prophage DNA insertion	none	Phage specific genes were found in REL606	none	tinyurl.com/ybokuag
7. Insertion,translocation and inversion	none	Prophage DNA insertion and translocation of nitrite reductase 2 genes	Phage specific genes found in DH10B	Translocation in DH10B is evident by dotplot. Moreover, the translocated genes in DH10B were found to be inverted. It could not be determined genes on which genomes were inverted as tranposon insertions were found in both genomes.	tinyurl.com/yaxlh7o http://tinyurl.com/y9cs6ft
8. Insertion and deletion	Transposon insertions and deletion of phenylacetic acid degradation genes	IS and Rac prophage DNA insertion	Phage specific genes found in DH10B. IS or transposon insertions in REL606 might have created direct repeats and facilitated excision of phenylacetic acid degradation genes.	Rac prophage DNA disrupted by transposon insertion in DH10B	tinyurl.com/yccbmsq
9a. Insertion	9a. none	9a. Insertion of IS5 sequence	9a. none	9a. none	9a. http://tinyurl.com/ylllc6u
9b. Insertion	9b. Insertion of ISI transposon	9b. none	9b. none	9b. none	9b. tinyurl.com/ygsqg2f
9c. Insertion	9c. ISI insertion	9c. Insertion of ABC transporter, flagella encoding genes and few other enzymes	9c. Inserted DNA segment in DH10B is bordered by direct repeats at both ends. 100% identity was found between the two repeats.	9c.DR indicates transposon insertion in DH10B.	9ctinyurl.com/yza4jy3 [2]
9d. Insertion	9d. IS2 insertion	9d. none	9d. none	9d. none	9d. tinyurl.com/ygfgtqy
10. Insertion and deletion	Bacteriophage DNA insertion and IS1 transposon insertion. Deletion of ~5 genes	Insertion of IS3	IS1 insertion at the site of deletion. Another IS1 insertion might have created direct repeats and facilitated deletion.	none	tinyurl.com/ykynub2
11. Insertion and deletion	IS1 insertion	CP4-57 prophage DNA insertion and possible deletion of ParB family protein and recombinase	Phage insertion in DH10B may have created pseudogene of ParB family protein genes and recombinase which later got deleted	Pseudogenes of yqa, yga and ypj indicated possible formation of pseudogenes of ParB and recombinase at some time prior to their deletion in DH10B.	tinyurl.com/yg7ybg4
12. Insertion, deletion and Inversion	IS1 insertion.	IS5 and IS10 transposon insertion. Inversion of ornithine decarboxylase, M-type protein and bifunctional prepilin peptidase/methylase. Deletion of saframycin synthetase, capsule related genes, bio-film formation genes, anti-toxin system and type II secretory apparatus genes.	Inversion in DH10B as evidenced by inverted repeats of IS10 transposon. Deletion of several genes in DH10B is evidenced by IS5 trans-activator transposase and presence of pseudogenes in DH10B,	Insertion of IS5 trans-activator transposase indicates possible deletion of several genes in DH10B. Also, no evidence of insertion in REL606 was found such as direct repeats.	tinyurl.com/yhyxgrq
13a. Deletion	13a. none	13a. Deletion of putative adhesin	13a. No direct repeats were found to indicate insertion of putative adhensin in REL606 therefore deletion in DH10B may have happened	13a.none	13a. tinyurl.com/yjojy53
13b. Insertion	13b. IS1 insertion and deletion of lipopolysaccharide genes	13b. none	13b. IS1 insertion in REL606 indicates that deletion may have occured by formation of directed repeats.	13b. IS1 insertion created pseudogene.	13b.http://tinyurl.com/yj2yg5s
13c. Insertion and deletion	13c. Insertion of IS30 transposon and several 'hypothetical protein" genes.	13c. none	13c. Insertion in REL606 is evidenced by direct repeats	13c. direct repeats were found in REL606 which indicates insertion of ShiA-like and TrbC-like genes.	13c. tinyurl.com/yjzdyum
14a. Insertion	14a. Insertion of several transposons and secondary glycine betaine transporter	14a.Insertion of several transposons. Insertion of Kple2 phage-like element	14a. Direct repeats bordering secondary glycine betaine transporter indicates its insertion	14a. none	14a. tinyurl.com/yzyvunx
14b. Insertion	14b. Insertion of ~15 genes	14b. Phage insertion. Transposon insertions	14b. Insertion in REL606 is evidenced by direct repeats flanking the DNA segment containing several genes.	14b.Phage-like genes were found in DH10B	14b. tinyurl.com/yly2b6u
14c. Deletion	14c. none	14c. Deletion of ~15 genes.	14c. Deletion in DH10B is evidenced by insertion of IS10R which may have facilitated excision of DNA by forming direct repeats	14c. Pseudogenes found at the site of deletion and IS10R insertion.	14c. tinyurl.com/yfhhsk6

@@ Line 56: / Line 56: @@
 To determine what the genes are in the inserion,just click on the gene models and their annotations will appear in a [[dialog box]] in [[GEvo]].  While many of the genes are annotated "hypothetical", several are annotated as phage genes.  This is likely a prophage.
+==Differences at an insertion site==
 Let us consider the ninth break in the dotplot. Run [http://tinyurl.com/yk9kt7e GEvo Analysis] for this break.