1. Sequencing Data Analysis
Debashis Sahoo
Department of Computer Science
CSE291 – H00 – Lecture 17

2. Sanger dideoxy sequencing--basic method
Single stranded DNA 3’ 5’ 5’ 3’
a) Anneal the primer

3. An automated sequencer
The output

4. Sequence output Computer calls Raw data
GNNTNNTGTGNCGGATACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCACCACCAC
CACCACCACCCCATGGGTATGAATAAGCAAAAGGTTTGTCCTGCTTGTGAATCTGCGGAACTTATTTATGATCCAGAAAG
GGGGGAAATAGTCTGTGCCAAGTGCGGTTATGTAATAGAAGAGAACATAATTGATATGGGTCCTAAGTGGCGTGCTTTTG
ATGCTTCTCAAAGGGAACGCAGGTCTAGAACTGGTGCACCAGAAAGTATTCTTCTTCATGACAAGGGGCTTTCAACTGCA
ATTGGAATTGACAGATCGCTTTCCGGATTAATGAGAGAGAAGATGTACCGTTTGAGGAAGTGGCANTCCANATTANGAGT
TAGTGATGCAGCANANAGGAACCTAGCTTTTGCCCTAAGTGAGTTGGATAGAATTNCTGCTCAGTTAAAACTTCCNNGAC
ATGTAGAGGAAGAAGCTGCAANGCTGNACANAGANGCAGNGNGANAGGGACTTATTNGANGCAGATCTATTGAGAGCGTT
ATGGCGGCANGTGTTTACCCTGCTTGTAGGTTATTAAAAGNTCCCGGGACTCTGGATGAGATTGCTGATATTGCTAGAGC

5. Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR)
The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA
A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

6. The three main steps of PCR
Step 1: Denature DNA
At 95C, the DNA is denatured (i.e. the two strands are separated)
Step 2: Primers Anneal
At 40C- 65C, the primers anneal (or bind to) their complementary sequences on the single strands of DNA
Step 3: DNA polymerase Extends the DNA chain
At 72C, DNA Polymerase extends the DNA chain by adding nucleotides to the 3’ ends of the primers.

7. PCR: Polymerase Chain Reaction
Step 1: denaturation
Step 2: annealing
Step 3: extension

8. PCR
PCR tubes
PCR C1000 Thermal Cycler

9. Denaturation of DNA
This occurs at 95 ºC mimicking the function of helicase in the cell.

10. Step 2 Annealing or Primers Binding
Reverse Primer
Forward Primer
Primers bind to the complimentary sequence on the target DNA. Primers are chosen such that one is complimentary to the one strand at one end of the target sequence and that the other is complimentary to the other strand at the other end of the target sequence.

11. Step 3 Extension or Primer Extension
DNA polymerase catalyzes the extension of the strand in the 5-3 direction, starting at the primers, attaching the appropriate nucleotide (A-T, C-G)

12. The next cycle will begin by denaturing the new DNA strands formed in the previous cycle

13. The Size of the DNA Fragment Produced in PCR is Dependent on the Primers
The PCR reaction will amplify the DNA section between the two primers.
If the DNA sequence is known, primers can be developed to amplify any piece of an organism’s DNA.
Forward primer
Reverse primer
Size of fragment that is amplified

14. FASTA
>SEQUENCE_1 MTEITAAMVKELRESTGAGMMDCKNALSETNGDFDKAVQLLREKGLGKAAKKADRLAAEG LVSVKVSDDFTIAAMRPSYLSYEDLDMTFVENEYKALVAELEKENEERRRLKDPNKPEHK IPQFASRKQLSDAILKEAEEKIKEELKAQGKPEKIWDNIIPGKMNSFIADNSQLDSKLTL MGQFYVMDDKKTVEQVIAEKEKEFGGKIKIVEFICFEVGEGLEKKTEDFAAEVAAQL >SEQUENCE_2 SATVSEINSETDFVAKNDQFIALTKDTTAHIQSNSLQSVEELHSSTINGVKFEEYLKSQI ATIGENLVVRRFATLKAGANGVVNGYIHTNGRVGVVIAAACDSAEVASKSRDLLRQICMH

15. FASTQ
@HWUSI-EAS466_0001:1:1:6:1464#0/1 CAAATGTCTATTTTNTCCGTCAATCTGTGAGTGNCA +HWUSI-EAS466_0001:1:1:6:1464#0/1 ACCTGGTCCTCTTTNAAGACGCGATGTGTCACGNTG +HWUSI-EAS466_0001:1:1:6:579#0/1 CGAATATCGTGACCNACCGCGGTACAATTGCATNCT +HWUSI-EAS466_0001:1:1:6:1050#0/1 a``aaaa`Y\T`aaBaa_^_``\`a```[O]__Ba`

16. The different types of BLAST
BLAST = Basic Local Alignment Search Tool
“The most popular data mining tool ever”
BLASTN DNA sequence vs. DNA sequence database
BLASTP protein sequence vs. protein sequence database
BLASTX DNA sequence translated in 6 reading frames
vs. protein sequence database
tBLASTX DNA sequence translated in 6 reading frames
vs. DNA sequence database translated in 6 frames

17. Steps to use Blast #1) Paste sequence here #2) Choose search set
(Either nucleotide collection or Protein Data Bank)
#4 push blast button
#3) select program to use

18. An example of aligning text strings
Raw Data ??? T C A T G C A T T G
2 matches, 0 gaps
T C A T G | | C A T T G
3 matches (2 end gaps)
T C A T G | | | C A T T G
4 matches, 1 insertion
T C A - T G | | | | C A T T G
T C A T - G | | | | C A T T G

19. Terminologies of sequence comparison
Sequence identity -- exactly the same Amino Acid or Nucleotide in the same position.
Sequence similarity -- Substitutions with similar chemical properties.
Sequence homology -- general term that indicates evolutionary relatedness among sequences; we usually measure of percentage identity of sequence homology
Pairwise alignment -- used to find the best-matching piecewise (local) or global alignments of two query sequences. Pairwise alignments can only be used between two sequences at a time.
Multiple sequence alignment -- try to align all of the sequences in a given query set.

20. Where are the coding regions?
TCAGCGAAGATGAGATAGTTTTTAAAGGTGGGATTTCCCCACCTTTAAAAAGCGAGAAGTCCCGGTTTTAAAGAGGAGTAAAATCCTCTTTTTCTAGCCCACTCAGGTGGTTTTTTTGGTTTTCGCTCCTTGCCGCATCTTCTGTGCCTTTGATGGCGGCTGGTTGGGGTGAAAGGCTGCATATTCCAGAATTTCAGACAGTAGATTGTTTTTGAAATCTTCCGTTTTATCGTTGACGAACTTAACCATCCTGTTGAAATCATCTTCCTTTGATACACCTTCAGGAAATGCCTTAGGAACTGATGTTTGGCTATCCAAGGCATCTTGCAATATCTGCACGATCTCCGAATTCATTGATCGCCCATTGGCCTTTGCTCTGGCGGCAACTGCGTCACGCATACCGTCAGGCATCCTAACTGTAAATCTCTCAATGAAAGCTGGATCTTCTTTTTCAGTCATCATCTTAAACCATAAAAATTTATACAAAACACACTAGCATCATATTGACATTACCCACAATGACATCATAATGGTGTCAGGCATCAAAATGATGTCATCATGACAAGGGGAAAGTAAATGCAAGATGTTCTCTATACAGGTCGTAAGAACGACAGCTTTCAGCTTCGTCTGCCTGAGCGAATGAAAGAAGAGATCCGTCGCATGGCAGAGATGGACGGCATTTCGATTAATTCTGCAATCGTGCAGCGCCTTGCTAAAAGCTTGCGTGAGGAAAGAGTTAATGGGCAGTAAAAACAGCGAAGCCCGGAAGTGTGGGGACACTAACCGGGCTTCTAATGTCAGTTACCTAGCGGGAAACCAACAATGACCAGTATAGCAATCTTTGAAGCAGTAAACACTATCTCTCTTCCATTCCACGGACAGAAGATCATAACTGCGATGGTGGCGGGTGTGGCGTATGTGGCAATGAAGCCCATCGTGGAAAACATCGGTTTAGACTGGAAGAGCCAGTATGCCAAGCTCGTTAGTCAGCGTGAAAAGTTCGGGTGTGGTGATATCACCATACCTACCAAAGGTGGTGTTCAGCAGATGCTTTGCATCCCTTTGAAGAAACTGAATGGATGGCTCTTCAGCATTAACCCAGCAAAAGTACGTGATGCAGTTCGTGAAGGTTTAATTCGCTATCAAGAAGAGTGTTTTACAGCTTTGCACGATTACTGGAGCAAAGGTGTTGCAACGAATCCCCGGACACCGAAGAAACAGGAAGACAAAAAGTCACGCTATCACGTTCGCGTTATTGTCTATGACAACCTGTTTGGTGGATGCGTTGAATTTCAGGGGCGTGCGGATACGTTTCGGGGGATTGCATCGGGTGTAGCAACCGATATGGGATTTAAGCCAACAGGATTTATCGAGCAGCCTTACGCTGTTGAAAAAATGAGGAAGGTCTACTGATTGGCGTATTGGAAGGCGCAAAAAGAAAAGCCAGCAGATGGGCTGCTGGCATTCATTGGGTATATGAACTTTCGGAGAACATATGAAGTCAATTATCAAGCATTTTGAGTTTAAGTCAAGTGAAGGGCATGTAGTGAGCCTTGAGGCTGCAAGCTTTAAAGGCAAGCCAGTTTTTTTAGCAATTGATTTGGCTAAGGCTCTCGGGTACTCAAATCCGTCA

21. Exon prediction in Eukaryotic DNA using Genescan: Net result is a protein sequence
GeneScan looks for start and stop codons, promoters, splice sites, polyA tails, provides statistics for coding potential

22. NGS sequencing pipeline

23. Sequencing steps Library preparation Library amplification
Parallel sequencing
Voelkerding KV et al., J Mol Diagn (2010) 12,

24. NGS Application Whole genome sequencing Whole exome sequencing
RNA sequencing
ChIP-seq/ChIP-exo
CLIP-seq
GRO-seq/PRO-seq
Bisulfite-Seq

25. Shyr D, Liu Q. Biol Proced Online. (2013)15,4
Patient
Technologies
Data Analysis
Integration and interpretation
point mutation
Small indels
Further understanding of cancer and clinical applications
Genomics
WGS, WES
Copy number variation
Functional effect of mutation
Structural variation
Differential expression
Transcriptomics
RNA-Seq
Network and pathway analysis
Gene fusion
Alternative splicing
RNA editing
Integrative analysis
Methylation
Epigenomics
Bisulfite-Seq
ChIP-Seq
Histone modification
Transcription Factor binding
Shyr D, Liu Q. Biol Proced Online. (2013)15,4