Analysis of your 16s RNA

DNA sequencing Most current sequencing projects use the chain termination method –Also known as Sanger sequencing, after its inventor Based on action of DNA polymerase –Adds nucleotides to complementary strand Requires template DNA and primer

Chain-termination sequencing Dideoxynucleotides stop synthesis –Chain terminators Included in amounts so as to terminate every time the base appears in the template Use four reactions –One for each base: A,C,G, and T 3’ ATCGGTGCATAGCTTGT 5’ 5’ TAGCCACGTATCGAACA* 3’ 5’ TAGCCACGTATCGAA* 3’ 5’ TAGCCACGTATCGA* 3’ 5’ TAGCCACGTA* 3’ 5’ TAGCCA* 3’ 5’ TA* 3’ Sequence reaction products Template

Sequence detection To detect products of sequencing reaction Include labeled nucleotides Formerly, radioactive labels were used Now fluorescent labels Use different fluorescent tag for each nucleotide Can run all four reactions in same lane TAGCCACGTATCGA A* TAGCCACGTATC* TAGCCACG* TAGCCACGT*

Sequence separation Terminated chains need to be separated Requires one-base-pair resolution –See difference between chains of X and X+1 base pairs Gel electrophoresis –Very thin gel –High voltage –Works with radioactive or fluorescent labels – + C A G T

Sequence reading of radioactively labeled reactions Radioactive labeled reactions –Gel dried –Placed on X-ray film Sequence read from bottom up Each lane is a different base ATCG + –

Capillary electrophoresis Newer automated sequencers use very thin capillary tubes Run all four fluorescently tagged reactions in same capillary Can have 96 capillaries running at the same time 96–well plate robotic arm and syringe 96 glass capillaries load bar

Sequence reading of fluorescently labeled reactions Fluorescently labeled reactions scanned by laser as a particular point is passed Color picked up by detector Output sent directly to computer

Summary of chain termination sequencing

Sequence databases What is a database? –An indexed set of records –Records retrieved using a query language –Database technology is well established Examples of sequence databases –GenBank Encompasses all publicly available protein and nucleotide sequences –Protein Data Bank Contains 3-D structures of proteins

The biological importance of sequence alignment Sequence alignments assess the degree of similarity between sequences Similar sequences suggest similar function –Proteins with similar sequences are likely to play similar biochemical roles –Regulatory DNA sequences that are similar will likely have similar roles in gene regulation Sequence similarity suggests evolutionary history –Fewer differences mean more recent divergence

Sequence alignment Sequence alignments search for matches between sequences Two broad classes of sequence alignments –Global –Local Alignment can be performed between two or more sequences QKESGPSSSYC VQQESGLVRTTC Global alignment Local alignment ESG

The algorithmic problem of aligning sequences Comparison of similar sequences of similar length is straightforward How does one deal with insertions and gaps that may hide true similarity? How does one interpret minimal similarity? –Are sequences actually related? –Is alignment by chance? QQESGPVRSTC QKGSYQEKGYC QQESGPVRSTC RQQEPVRSTC QQESGPVRSTC QKESGPSRSYC

Methods of sequence alignment Graphical methods Dynamic-programming methods Heuristic methods

A practical example of sequence alignment

BLAST results

Detailed BLAST results

A pairwise alignment with MASH-1 HASH-2, a human homolog of MASH-1 –“+” indicates conservative amino acid substitution –“–” indicates gap/insertion –XXXX… shows areas of low complexity