Analysis of your 16s RNA

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