Transcript Shotgun Sequencing

Max Bachour
Jessica Chen
Shotgun or 454 sequencing
•High throughput sequencing technique that can
collect a large amount of data at a fast rate.
•Works by partially digesting a genome or big
strand of DNA into small overlapping fragments
•These small fragments are sequenced and
fragments that overlap are matched together.
Steps Behind 454 sequencing
a. The genome is fragmented and the
fragments are denatured.
b. Fragments are amplified and
assigned to beads. One fragment
per one microbead.
c. Each bead is placed in the wells of
a fiber optic slide.
d. Packing beads placed in all the
wells.
Steps Behind 454 sequencing
• Solution of one nucleoside is
flooded onto tray.
• If base added is next in the
sequence, it will be added to
the single stranded DNA on the
bead.
• When a nucleoside is added to
DNA, 2 phosphates are given
out
• Enzymes in packing beads
convert phosphate groups to
ATP and then the ATP to light
energy.
Steps Behind 454 sequencing
• Computer and camera detect
light in a certain well as a
certain base is added to the
tray.
• Base is washed off and
process is repeated with
another base.
• End product is large amount of
fragments sequenced.
Genome Sequence
Analysis
Contig Assembly
Identifying open reading frames (ORF) using gene
prediction programs
What is the initial problem with assembly?
Sequenced
fragmented
DNA
CONTIG 1
Incorrectly
Assembled
DNA Sequence
CONTIG 2
How is this problem solved?
Sequenced
fragmented
DNA
Masked DNA
Sequence
Assembled
DNA Sequence
CONTIG 1
CONTIG 3 CONTIG 4
CONTIG 2
CONTIG 5
How do we identify genes?
1) Use gene prediction programs (Fgenesh,
Genscan, Genemark) to determine
potential genes; also determine any
repeat sequences
 Enter contig
2) Which of the predicted genes are most
likely existing genes?
 Use BLAST
How do we use BLAST?
 tblastn all predicted genes against
an EST database (ESTDB)
Why ESTDB? – record of all known/identified
mRNA (cDNA library)
Why tblastn? -- amino acid sequence more likely
to be conserved
 use blastn and blastp
-blastp: determine expression of gene
Analyzing BLAST data
Gene 1:
Protein sequence:
• Critical data:
– e-value
– %match
– EST source
MFVVQYLGSSRSWTSCSHSSKPGVDSRGRAEPHLAVGRSSLLGRVQTGLKGGGMKDSDLT
GDSSLARANQSMGICKSEGTVDRRLKSQVSQLLLGLLLIRLEGLLATCMTGPHGDAGAGS
THK
>gb|FC457105.1| UCRVU04_CCNI646_g1 Cowpea 524B Mixed Tissue and Conditions cDNA
Library UCRVU04-1 Vigna unguiculata cDNA clone CCNI646, mRNA
sequence.
Length=807
Score = 215 bits (548), Expect(2) = 2e-55, Method: Compositional matrix adjust.
Identities = 110/112 (98%), Positives = 110/112 (98%), Gaps = 0/112 (0%)
Frame = -1
Query 12 SWTSCSHSSKPGVDSRGRAEPHLAVGRSSLLGRVQTGLKGGGMKDSDLTGDSSLARANQS
71
SWTSCSHS KPGVDSRGRAEPHLAVGRSSLLGRVQTGLKGGGMKDSDLTGDSSLARANQS
Sbjct 438 SWTSCSHS*KPGVDSRGRAEPHLAVGRSSLLGRVQTGLKGGGMKDSDLTGDSSLARANQS
259
Advantages and Disadvantages
• Fast sequencing at a high volume
• Cheap compared to other methods
• Much higher coverage protection
• Repetitive sequences can disrupt
computer program into thinking that
unrelated sequences are in fact
connected.
• More prone to error and missing
sequences
Drastically changed
genomics in a very
short amount of
time