Max Bachour Jessica Chen Shotgun or 454 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 à 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 m. RNA (c. DNA 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|FC 457105. 1| UCRVU 04_CCNI 646_g 1 Cowpea 524 B Mixed Tissue and Conditions c. DNA Library UCRVU 04 -1 Vigna unguiculata c. DNA clone CCNI 646, m. RNA sequence. Length=807 Score = 215 bits (548), Expect(2) = 2 e-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