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Analysis of your 16s RNA

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.

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Analysis of your 16s RNA

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  1. Analysis of your 16s RNA

  2. 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

  3. 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’ 5’ TAGCCACGTATCGAACA* 3’ 5’ TAGCCACGTATCGAA* 3’ 5’ TAGCCACGTATCGA* 3’ 5’ TAGCCACGTA* 3’ 5’ TAGCCA* 3’ 5’ TA* 3’ Sequence reaction products

  4. 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 TAGCCACGTATCGAA* TAGCCACGTATC* TAGCCACG* TAGCCACGT*

  5. 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 C A G T

  6. A T C G 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 +

  7. 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

  8. 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

  9. Summary of chain termination sequencing

  10. 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

  11. 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

  12. 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

  13. 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? QKESGPSRSYC QQESGPVRSTC RQQEPVRSTC QQESGPVRSTC QKGSYQEKGYC QQESGPVRSTC

  14. Methods of sequence alignment • Graphical methods • Dynamic-programming methods • Heuristic methods

  15. A practical example of sequence alignment

  16. BLAST results

  17. Detailed BLAST results

  18. 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

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