Transcript A Flexible Approach to Implement Genomic

Genomics Education Partnership: a flexible approach to implement Genomic teachings and
research in the classroom
Matthew W. Wadsworth and Consuelo J. Alvarez, Department of Biological and Environmental Sciences,
Longwood University, Farmville, VA 23901
INTRODUCTION
ANNOTATION
The Genomics Education Partnership (GEP) has afforded students at Longwood University the opportunity to work with finishing sequencing and annotation research projects
that are of scientific significance. The project focuses on many closely related Drosophila species (Fig. 1). The purpose of the project is to assess the differences between the dot
chromosome of D. melanogaster, which is largely heterochromatin, and others species such as D. mojavensis, D. erecta, and D. virilis that are composed mainly of euchromatin.
The long-term goal of the project is to use comparative genomics to discover the evolutionary cause of the relatively recent transition from a euchromatic to heterochromatic dot
chromosome.
Each student begins by selecting a finishing or annotation project from an online database. Finishing the DNA sequence is the first step. The projects were compiled by the
Genome Sequencing Center (GSC) at Washington University, St. Louis, for use by students. Genomes enter the GSC as BAC or fosmid libraries from which clones to be
sequenced are chosen. The GSC then prepares approximately 2 kb libraries from each clone that are then shotgun sequenced (Fig. 2). When these DNA fragments are then
pieced together using Phred/Phrap there can be a wide variety of problems with the sequence, such as gaps or low quality areas that then must be corrected by the finisher.
Annotation is the process of locating genes and other relative sequences within the finished DNA sequence. This process requires the use of various online gene databases and
results in specific gene locations with exact exon/intron boundaries which can then be used for comparative analysis.
The process of mapping the location of genes and various other relevant sequences within a finished DNA sequence
GOALS: To find genes, its functions and coding proteins as well as to delimit exon-intron boundaries, and uncover isoforms and orthologs
PROCEDURE:
* Check basic information from various gene finding programs such as Genscan and GeneID (Fig. 7a-b)
* Mask the repetitious sequences that range from simple poly-A repeats to repetitious elements of over 50 base pairs
* Search a database containing matches to known gene sequences; use NCBI’s Basic Local Alignment Search Tool (BLAST) vs. D. melanogaster (Fig. 8)
* Map the exon/intron boundaries and check its accuracy with the Gene Model Checker program (provided by GEP)
* Note any sequences that are out of the ordinary
* Submit the project (Fig. 9)
Figure 1. Phylogenetic tree for Drosophila species showing that the D. melanogaster
subgroup has evolved more recently than D. virlis, prompting scientists to investigate the
reason for a change from a euchromatic to heterochromatic dot chromosome.
Figure 2. Genome shotgun sequencing pathway indicating that gaps in
the sequence of the assembly can form where no fosmids are recorded
across a given area.
FINISHING
A multi-step process used to piece together a complete and flawless DNA sequence
GOALS:
To eliminate any gaps, correct and improve low-quality regions, high-quality discrepancies between bases,
single subclones, or single chemistry for sequencing reads
PROCEDURE:
* Select DNA fosmids of approximately 40 kb in length from an online database
* Analyze these sequences by using the programs Consed and Phred/Phrap
* Look at the gaps present in the fosmid and the overall quality of the sequence (Fig. 3)
* Correct high-quality discrepancies between base pairs when enough evidence from other reads is present (Fig. 4)
* Call for reads to solve more complex problems such as gaps or low-quality areas that have no relevant data present (Fig. 5)
* Check for bacterial contaminants to be present with “BLAST”
* Continue to annotation with the final finishing product is complete and has a high-quality DNA in comparison to the consensus (Fig. 6)
Figure 3. A screenshot from the program Consed of the original assembly view of finishing project 120-D14
is shown. The DNA contig labeled 3 is a sequence approximately 29 kb in length, while contig 2 is roughly
12 kb in length. One of the primary duties of a finisher is to bridge the gap between these two contigs in
order to create a single continuous contig roughly 41 kb long. The green lines spanning the assembly
represent the amount of coverage over the area as well as relative quality of the data present. The triangle
lines represent forward and reverse pairs, and the orange and black boxes show tandem and inverse
repeats. Additional reads may be required to increase the quality of certain areas to an acceptable level.
Figure 4. An example of a
high quality discrepancy
between base pairs is
given in this figure. Each
row represents a separate
read over the target area.
While all reads are of equal
quality at that particular
base (13,533 of the
consensus
sequence),
some call it as a cytosine
and others as a thymine.
This discrepancy is an
example
of
a
polymorphism and can not
be manually corrected by
the finisher. Instead the
finisher tags the area so
that more detailed research
can be conducted later on.
Figure 7a. Visual representation of the Genscan program predicted
genes from the annotation project containing fosmid13. Genscan
has predicted 5 total genes within the fosmid, three in the plus
reading frame and two in the minus reading frame.
Figure 7b. A more specific read-out of
predicted genes provided by the Genscan
program is described. Contained within are
predicted exon/intron boundaries are shown
as well as relevant sequences such as the
promoter region and poly-A tails.
Figure 9. A summary
of the actual genes
located
within
fosmid13 is shown.
Notice that five genes
were
originally
predicted (Fig. 7),
however, only two
genes were actually
found to be present
within fosmid13.
Figure 8. Blastn results indicating a section of a suspected gene
sequence within fosmid13 after being run through a D.
melanogaster gene database. The query sequence represents the
D. melanogaster gene location within the chromosome, and the
subject sequence is the suspected D. erecta gene. The sequences
are 96% matches to one another, with an example of a mismatched
base pair circled in red.
CONCLUSION
Figure 5. Two separate sets of reads called for project 120-D14 are described. The column entitled Oligo
Sequence shows the selected DNA primer sequence from which the read will originate from. Primers are selected
70-100 bases from the problem area and are oriented to span the region in question. Through use of these reads
the two contigs were joined and all low-quality, single subclone/single chemistry areas were remedied.
The implementation of the Genomics Education Partnership at Longwood University was successful. At Longwood University a total of five
annotation and four finishing projects were completed and submitted during my two-semester involvement with this project. The finishing of D.
mojavensis and annotation of D. virilis has since been completed. Although, we did start with the annotation of D. erecta, this current year, the GEP had
added to its research new drosophila species. Thus, the target organism for finishing is D. grimshawi while for annotation is D. mojavensis as well as to
complete the remaining fosmids of D. erecta. The data obtained through the completion of these projects will go a long way in assisting upper-level
researchers in determining the evolutionary transition from a euchromatic to heterochromatic dot chromosome. The GEP provides a unique educational
experience, allowing students to be involved in a project that requires collaboration with other students and faculty spread across the country.
REFERENCES
Figure 6. Assembly view of project 120-D14, obtained upon project completion, showing that the initial gap has
been bridged, and all other errors corrected. The fosmid will continue on to annotation from this point.
GEP Homepage: http://gep.wustl.edu/
NCBI BLAST Search Engine: http://blast.ncbi.nlm.nih.gov/Blast.cgi
FlyBase: http://flybase.org/
UCSC Genome Browser: http://genome.ucsc.edu/
RepeatMasker: http://www.repeatmasker.org/
Genscan: http://www.genscan.com/
ACKNOWLEDGMENTS
GEP Program Director: Sarah C.R. Elgin
Technical Director: Chris Shaffer
Chief Technical/Teaching Assistant: Wilson Leung
Sponsored by Washington University at St. Louis and HHMI
GEP members and partner 06-07-08 and their students and institutions
Biology 425 students, spring class 2008 at Longwood University