ABC-INT-Genome annotation

Integrator Unit: Genome annotation

(Integrator unit: annotate sequences in a genome)

 

Abstract:

This page assesses the learning units for data management and sequence analysis of genomic sequence data.

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Deliverables:

Integrator unit: Deliverables can be submitted for course marks. See below for details.

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Prerequisites:
This unit builds on material covered in the following prerequisite units:

• BIN-FUNC-Annotation (Function Annotation)
• BIN-Genome-Browsers (Genome Browsers)

 

 

 

Evaluation

This "Integrator Unit" should be submitted for evaluation for a maximum of 13 marks if one of the written deliverables is chosen, resp. 24 marks if you choose this for your oral test.

Please note the evaluation types that are available as options for this unit.

Be mindful of the Marking rubrics.

If this is submitted for your oral test, please read the Oral test instructions before you begin.

If your submission includes R code, please read the Code submission instructions before you begin.

Once you have chosen an option ...

1. Create a new page on the student Wiki as a subpage of your User Page.
2. Put all of your writing to submit on this one page.
3. When you are done with everything, go to the Quercus Assignments page and open the appropriate Integrator Unit assignment. Paste the URL of your Wiki page into the form, and click on Submit Assignment.

Do not change your Wiki page after you have submitted your assignment, until it has been graded.

 

Report option

• Work through the tasks described below.
• Document your results in a short technical report on a subpage of your User page on the Student Wiki. Describe your methods in your report to an appropriate level of detail that your analysis can be exactly reproduced. If you write R-code, include the code in your report;
• When you are done, submit the link to your page via Quercus as described above.

 

 

Literature research option

This option requires that a primary publication is available for the MYSPE genome sequence; if there is none, this option is not available.

• Write a report on the annotation methodology that was used for the MYSPE genome. Note: this is not a review, but a report. Think of a "whitepaper", not a publication. Write to a specialist technical audience - imagine collaborators who want to use the same methods - and be specific to provide actionable information (links, instructions, resource requirements ...).
• Include a sketch of the workflow;
• write your report on a subpage of your User page of the Student Wiki;
• make sure that you have included all references and citations.
• the level of detail should be sufficient to allow an undergraduate project student to reproduce the analysis.

• When you are done, submit the link to your page via Quercus as described above.

 

Oral test option

• Work through the tasks described below. Remember to document your work in your journal, but there is no need to format this specially as a report.
• Describe your methods in your report to an appropriate level of detail that your analysis can be exactly reproduced. If you write R-code, include the code in your report;
• You should be prepared to explain and interpret your findings in the test.
• Note that the work must be completed before your actual test date.

 

Contents

 

Scenario

 

You know that MYSPE has an Mbp1 orthologue. Key questions of functional genome annotation could be: does it work in the same way in MYSPE as in yeast? Does it have the same target genes? Is it regulated by orthologues to other yeast genes that imply the same feedback mechanisms and genetic regulatory circuits? Here we will try to deduce just one part of such questions: is the binding motif for Mbp1 conserved? If that is the case, we could automate the task to find genes that are potentially regulated by MBP1_MYSPE, if not, we would need to pursue a different strategy of binding site discovery.

Here is how we assess the conservation of the Mbp1 DNA binding motif in MYSPE, working from the orthologue of CDC6, a pre-replicative complex component that is one of Mbp1's target genes:

• Find the MYSPE orthologue for yeast CDC6 and document your search and result.
• Fetch a contiguous segment of genome sequence: 500 nucleotides of upstream genome sequence plus the first thirty nucleotides of coding sequence. Use a method that will work at scale, given chromosomal coordinates: a link to the NCBI genome record as in the example below will be fine, similar links could be generated from UCSC or ensembl resources, or with a few lines of biomart:: code. Manual selection and copy/paste from a sequence database record is not acceptable for this assignment.
• Demonstrate that this is the correct sequence by showing and annotating the 530 nucleotides in your submission (refer to the example below for contents and formatting). Add the translation of the first 10 codons of the CDC6-orthologue to your annotation. Make sure that you are showing the correct reverse complement in case your orthologue is transcribed from the (-)-strand!
• In your submission:
  • You must include the correct database identifiers on which you are basing your analysis, linked to their respective sources;;
  • There must be a link to the genome sequence source (with chromosomal coordinates) and it must span exactly 530 nucleotides;
  • There must be a link to the protein sequence and it must start with the translated amino acids;
  • The FASTA header of the downloaded nucleotide sequence must be included;
  • Upstream sequence must be listed in ten lines of 50 nucleotides each;
  • There must be ten codons on the next line;
  • The first of the ten codons must be the CDC6-orthologue start codon, and the translation must be shown;
  • The motifs you find and discuss must be indicated in the annotated sequence listing.
• The yeast Mbp1 canonical binding site is defined by the regular expression "[AT]CGCG[AT]". (Please review RPR-RegEx if you are not sure about the meaning of "[" and "." in a regular expression.)
• Are there CGCG motifs present in your nucleotide sequence?
• Identify them using a regular expression search. Refer to RPR-RegEx to review the use of gregexpr() and regmatches(). The following code-sample may get you started:

patt <- "..CGCG.."
m <- gregexpr(patt, mySeq)
regmatches(mySeq, m)[[1]]

• Are there [AT]CGCG or CGCG[AT] motifs? What about [AT]CGCG[AT]?
• Where are the motifs located? Do they cluster? Are they arranged in a similar way as the yeast binding sites that you visited at UCSC?
• Interpret your finding by contrasting your observation to the situation with yeast. Does your analysis support or refute the idea that the CDC-orthologue in MYSPE is regulated by a transcription factor with the same DNA sequence binding specificity as MBP1_SACCE? Can you make an argument whether that transcription factor could or could not be the Mbp1-orthologue in MYSPE?

 

Sample annotation

(Demonstrating the required level of detail for a valid submission)

• MYSPE: Sporothrix Schenckii (1397361)
• CDC6 (NP_012341) orthologue (by RBM): XP_016592126
  

(coverage: 72%; E: 4e-27; ID: 26.08%)
Reverse search in taxID:4932 finds NP_012341 as the top hit.

• Protein FASTA of XP_016592126
• Translation-start ATG: range 1255377 .. 1255379
• Link to Genomic sequence (FASTA) (Range: 1254877..1255406)

>ref|NW_015971139.1|:1254877-1255406 Sporothrix schenckii 1099-18 chromosome Unknown Cont38, whole genome shotgun sequence

  5'-TCCACCAAACTAGTCGGGCGAGCTGAACTATGTCGTCCGCCATTTAAAGC

     CCACTGTACGAATAGCGCAATACTGTAGACGACCGCACAGTGTATCTGTG

     GCTAGTGTGCAAGCACGCGCCACGGCAGCTGGGCGGGTCTGGGGTCAATC
                   =====x
     CTCCCACGTACGCGTAAAACCGCCAACGCGTCCAGCAATGGCAGGGGTAA
              ======
     GTCAGTCGCGCTTTCTTCGCGTAAAGTGGTTCCTCTATTTGGCGCGCGCT
          =====x
     TCCTCATTAAATCTTGTACCTCCCTTGGCCACCATCTTGAACTTTCCTTC

     GTGCTTTCCACGTTTGACTTCATTCCCTGTTACTTCCATTTTGTCCATTC

     TTGCGACTGTCTATTCTTTCTTTGCGAGCATCTACGCATCTATCCATCGT

     TCTTTCCGTTGTATGCATCTACGTCGCTGTTCTTGCCATTGCTTTACCCC

     TTTCTTTAAACCCTTCCTCCTTTGCTCTTTCCTCACCACACACTACAAAC

     ATG GTT GCT TCC TCG CTC GGA AAG CGG ATC.....      -3'
      M   V   A   S   S   L   G   K   R   I   ...

Notes

 

About ...
 
Author:

Boris Steipe <boris.steipe@utoronto.ca>

Created:

2017-08-05

Modified:

2020-10-01

Version:

1.1

Version history:

• 1.1 2020 Updates; add example annotated sequence; sequence fetch must not be copy/paste.
• 1.0.1 Capitalize CDC6
• 1.0 First live version
• 0.1 First stub

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