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

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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.

Webpage of SynMap

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

  1. All protein coding regions (CDS)) are extracted from each genome
  2. These sequences are blasted against each other to identify putative homologous gene pairs
  3. 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

Webpage of 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.

To determine identity of genes, click on individual genes and its annotation will 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

Using side bars zoom in on a region.

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.

Putative insertion in DH10B containing many flagellar genes.

This is a different type of insertion event. In REL606 there is a single gene insertion, while in DH10B there is a many gene insertion at the same genomic position. In REL606, the gene is boarded by black-blue boxes, which is how annotated repeated sequences are visualized in CoGe. This gene is an IS1 transposon, a class of DNA elements that move around a genome. The DH10B insertion contains a number of flagellar. These regions present some different possible evolutionary scenarios:

  1. Deletion in REL606: Perhaps two IS1 transposons landed in REL606 are were oriented in the same direction. This could provide direct repeat sequences needed for non-homologous recombination to remove the intervening sequence which included the flagellar genes
  2. Insertion in HD10B: Perhaps the flagellar genes were transferred in and replaced an IS1 element by hijacking its transposition machinery
  3. Insertion in both: Perhaps they are both new insertional events.

Since the sequence at this position is not overlapping between the regions, we can investigate this by adding a second copy of each region to the analysis, and looking for repeat sequences boarding these putative insertions. There are two ways to do this:

Adding and resizing
  1. adding new sequences:
    1. Click on "Add sequence" in GEvo
    2. Copy and paste the gene name into the "name" box. Do this for each region.
    3. Zoom in on the displayed sequences to use sequence right around the insertion and copy these positions in to the newly added sequences.
Adding and resizing
  1. Merge two analysis:
    1. Zoom in on region
    2. Run analysis
    3. Copy link from analysis into merge box and press merge

The order of the sequence can be changed by dragging the sequence submission boxes around relative to one another. Also, the alignment algorithm should be changed to blastn instead of blastz. Blastn is more sensitive than blastz for finding small regions of sequence similarity. You can use this link to generate the results of this 4-way analysis: http://tinyurl.com/y9pgzvl .

4-way GEvo analysis including self-self comparisons. http://tinyurl.com/y9pgzvl

The results from this 4-way analysis do not show any direct sequence repeats at the ends of either insertion. This means that there is not evidence that region in DH10B was inserted recently. Of the possible evolutionary scenarios, it is most likely that this region was deleted in REL606, probably as the result of the IS1 element, perhaps by the insertion of two of these elements followed by deletion of the intervening sequence.

Phage insertion

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

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.
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