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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 Template 3’ ATCGGTGCATAGCTTGT 5’ Sequence reaction products 5’ TAGCCACGTATCGAACA* 3’ 5’ TAGCCACGTATCGAA* 3’ 5’ TAGCCACGTATCGA* 3’ 5’ TAGCCACGTA* 3’ 5’ TAGCCA* 3’ 5’ TA* 3’ 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 TAGCCACGTATCGAA* 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 CAGTCAGT + Sequence reading of radioactively labeledA reactions T C G • Radioactive labeled reactions – – Gel dried – Placed on X-ray film • Sequence read from bottom up • Each lane is a different base + 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 robotic arm and syringe 96 glass capillaries 96–well plate 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 ESG ESG Local alignment 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? QKESGPSRSYC QQESGPVRSTC RQQEPVRSTC QQESGPVRSTC QKGSYQEKGYC QQESGPVRSTC 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