1. Today HK (TOPO)TA-cloning *.ppt NGS Illumina GALAXY seminar
Sequence editing and BLAST data quality, improve, identity of snails

3. SNAIL AND PARASITES BIOLOGY
DNA
“identity, possibilities”
phylogenetics
CTAB/DNAzol
CTAB/DNAzol
gel electrophoresis
nanodrop spec
Illumina (full)
genome sequencing
PCR rDNA/mito
Qubit Fluorometry
Covaris fragmentation
Ampure (fragment collection)
Kapa DNA library preparation kit
Pippin size selection
QC Bioanalyzer, Qubit, qPCR
Illumina run
TA cloning, B/W screening
electrophoresis
Qiagen plasmid extraction Restriction digests
direct sequencing
M13 sequencing
Sequence ID (BLAST)
editing
Primer design, walking
Galaxy
QC Data file
(MT) genome assembly
Mitos, manual annotation
Gene annotation
Phylogenetics
GenBank submission

5. BECAUSE OF TEMPLATE INDEPENDENT 3’ A-ADDITION TO AMPLICON
MORE EFFICIENT THAN LIGASE,
“OOPS”

6. HOW DOES IT WORK

7. IF Alpha complementation, BLUE/WHITE screening MCS
(alpha peptide)
One-shot E. coli have a non functional mutant lacZ gene with deleted sequence (i.e. lacZΔM15)
MCS
The plasmid
Encodes LacZ (alpha peptide) IF
not interrupted by an insert

8. Growth on selective media (Kanamycin 100mg/ml) Metabolize X-gal
(carbohydrate -> blue) R
antibiotics resistance
LacZ-a
LacZ
insert (!)

9. antibiotics resistance LacZ-a insert (!)
Growth on selective media
(Kanamycin 100mg/ml)
Metabolize X-gal to
screen for insert
(carbohydrate -> blue) NO
YES
YES R
antibiotics resistance
LacZ-a
insert (!)

10. antibiotics resistance LacZ LacZ-a insert (!)
Growth on selective media
(Kanamycin 100mg/ml)
Metabolize X-gal to
screen for insert
(carbohydrate -> blue) NO
(-)
YES
YES
YES NO R
antibiotics resistance
LacZ
LacZ-a
insert (!)

11. antibiotics resistance LacZ LacZ-a insert (!)
Growth on selective media
(Kanamycin 100mg/ml)
Metabolize X-gal to
screen for insert
(carbohydrate -> blue) NO
(-)
YES
YES
YES NO R
antibiotics resistance
LacZ
LacZ-a
insert (!)

12. 1) Do 10 minutes
7) Spread 10 on
one plate and 100 on another

13. SHARE COLONIES IF NEEDED
Friday
Each group Select 4 white colonies, sample by “scraping” with yellow tip
Eject yellow tip with bacteria in 2.5 ml LB, 100mg/ml kanamycin
SHARE COLONIES IF NEEDED
OR….?
NO colonies, troubleshoot, repeat!

14. *.ppt

15. NextSeq Illumina genome sequencing NEEDs
P3S: Physid snail
Strigeid cercariae
A: Ancylid, no parasites observed
Total DNA minus RNA
Illumina whole genome sequencing
Qubit Fluorometry
Covaris fragmentation
Ampure (fragment collection)
Kapa DNA library preparation kit
Pippin size selection
QC Bioanalyzer, Qubit, qPCR
Illumina run
P3S NGS
A NGS
NextSeq Illumina genome sequencing NEEDs
ng DNA for Kapa Hyper prep kit

16. SEQUENCING by
EXTENSION
versus
SYNTHESIS ?

17. 130 million, 2 x 150 (300nt) Paired End Reads
NextSeq Illumina run,
130 million, 2 x 150 (300nt) Paired End Reads
Result: ≤ 3,900,000,000 nucleotides (inspect all 365/24/60/60 = ~123 years)
Sequence quality: Trimming bad sequence
+ Filtering to remove adaptors, barcodes
Assembly

18. Bring your
LAPTOP
Use W7
LAPTOP
Dr LiJing Bu

19. DNA SANGER SEQUENCING
“*.ABI”
Sequence analysis? 19

22. Or copy/paste sequence to BLAST window(

23. BLAST IDENTIFICATION (RAW)
16SF R COIF R
P1E 1,2
7,8
L1S 3,4
9,10
P2E 5,6
11,12

24. - strand antisense non-coding
sequencing 5’
primerF
+ strand sense coding
primerR A 3’ 3’ A
primerF
- strand antisense non-coding
primerR 5’ 5’
ATCG
sense
GGAA A 3’
NEED TO KNOW
WHY SEQUENCE THIS? 3’ A
TACG
antisense
CCTT 5’ 24

25. sequencing forward 5’ + strand sense coding A 3’ 3’ A
primerF
+ strand sense coding
primerR A 3’ 3’ A
primerF
- strand antisense non-coding
primerR 5’ 5’
ATCG
sense
GGAA A 3’
Forward primer sequencing reaction 5’
ATCG 3’ 3’ A
TACG
- strand antisense non-coding
CCTT 5’ 25

26. sequencing results 5’ + strand sense coding A 3’ 3’ A
primerF
+ strand sense coding
primerR A 3’ 3’ A
primerF
- strand antisense non-coding
primerR 5’
Forward primer sequencing reaction
generates sense strand 5’
ATCG 3’
ggaa 3’ A
TACG
- strand antisense non-coding
CCTT 5’ 26

27. - strand antisense non-coding
sequencing reverse 5’
primerF
+ strand sense coding
primerR A 3’ 3’ A
primerF
- strand antisense non-coding
primerR 5’ 5’
ATCG
sense
GGAA A 3’ 3’ A
TACG
antisense
CCTT 5’ 27

28. - strand antisense non-coding
sequencing reverse 5’
ATCG
+ STRAND SENSE CODING
GGAA A 3’ 3’ A
tagc
- strand antisense non-coding
cctt 5’
Reverse primer sequencing reaction 5’
ATCG
SENSE
GGAA A 3’ 3’
CCTT 5’ 3’ A
TACG
antisense
CCTT 5’ 28

29. - strand antisense non-coding
sequencing results 5’
ATCG
+ STRAND SENSE CODING
GGAA A 3’ 3’ A
tagc
- strand antisense non-coding
cctt 5’
Reverse primer sequencing reaction 5’
ATCG
SENSE
GGAA A 3’
tagc
generates - strand 3’
CCTT 5’ 29

30. - strand antisense non-coding
sequencing results 5’
ATCG
+ STRAND SENSE CODING
GGAA A 3’ 3’ A
tagc
- strand antisense non-coding
cctt 5’
Reverse primer sequencing reaction 5’
ATCG
SENSE
GGAA A 3’
tagc
generates - strand 3’
CCTT 5’
ABI File generated 5’-3’
Sequence is reverse complement of coding sequence ! 30

31. Forward primer sequencing reaction
sequencing generates + strand 5’
ATCG 3’
ggaa
Reverse primer sequencing reaction
tagc
generates - strand 3’
CCTT 5’
sequencing generates + strand 5’
ATCG 3’
ggaa
reverse complement yields + (coding) sequence
two confirmatory sets of sequence data

32. - strand antisense non-coding
sequencing results 5’
ATCG
+ STRAND SENSE CODING
GGAA A 3’ 3’ A
tagc
- strand antisense non-coding
cctt 5’
Reverse primer sequencing reaction 5’
ATCG
SENSE
GGAA A 3’
tagc
generates - strand 3’
CCTT 5’
ABI File generated 5’-3’ 5’
TTCC 3’
dnarts - setareneg
cgat
Sequence is reverse complement of coding sequence ! 32

33. sequencing generates + strand
Forward primer sequencing reaction
sequencing generates + strand 5’
ATCG 3’
ggaa
dye blobs
Reverse primer sequencing reaction
tagc
generates - strand 3’
CCTT 5’
dye blobs

34. Sequence editing (H8)
Full length of amplicon? Look for other primer
Do we have all information? Edit, compare replicate reactions
Compare computationally to other sequences BLAST
AND Phylogenetic analysis for “best” identification

35. So full length?
Abrupt end
Primer?
Same for other sequencing reaction

36. ?
LCO1490: 5'-GGT CAA CAA ATC ATA AAG ATA TTG G -3’
HC02198: 5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’
Primer is reverse complement at end of sequence run

37. Sequence reverse complemented
LCO1490: 5'-GGT CAA CAA ATC ATA AAG ATA TTG G -3’
HC02198: 5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’
Primer is reverse complement at end of seqeunce run

38. Sequence reverse complemented
LCO1490: 5'-GGT CAA CAA ATC ATA AAG ATA TTG G -3’
HC02198: 5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’
Primer is reverse complement at end of sequence run

39. Fix “N” calls: what are these?
Stay at regular spacing distance

40. Fix “N” calls: what are these?
Stay at regular spacing distance

41. Dye Blobs?
Edit under the blob

42. A G C G T N A A NNNN
Dye Blobs?
Edit under the blob

43. A G C G T N A A NNNN
T T G A C G A G C A T T T T T A
Dye Blobs?
Edit under the blob

44. BLAST

45. BLAST

46. Need to do phylogenetics.
Pseudosuccinea columella mitochondrial COI gene for cytochrome oxidase subunit I, partial cds, isolate: Pc.2
Sequence ID: gi| |LC Length: 667Number of Matches: 2
Related Information
Range 1: 132 to 656GenBankGraphics
Next Match
Previous Match
Alignment statistics for match #1 Score Expect Identities Gaps Strand
569 bits(630) 2e /525(82%) 0/525(0%) Plus/Plus
Query TTTTGTTATAAtttttttCATANTTATACCAATAATANTTGGAGGGTNNGGAAATTGAAT 185
|||||||||||||||||||||| |||||||||||||| ||||||||| |||||||||||
Sbjct TTTTGTTATAATTTTTTTCATAGTTATACCAATAATAATTGGAGGGTTTGGAAATTGAAT 191
Query AGTTCCNCTTCTCATTGGNGCTCCNNATATAANATTTCCTCGNATAAATAATATANNANN 245
|||||| ||||||||||| ||||| |||||| ||||||||| |||||||||||| |
Sbjct AGTTCCACTTCTCATTGGTGCTCCAGATATAAGATTTCCTCGTATAAATAATATAAGATT 251
Query TTGATTACTACCACCTTCNNTTATTCTCTTACTTTGNTCTNGAATANNANAAGGTGGGGN 305
|||||||||||||||||| |||||||||||||||| ||| ||||| | |||||||||
Sbjct TTGATTACTACCACCTTCGTTTATTCTCTTACTTTGCTCTAGAATAGTAGAAGGTGGGGT 311
Query AGGNACTGGATGAACAGTTTACCCACCATTGANTGGACCTATTGCTCATGGNGGATCTTC 365
||| |||||||||||||||||||||||||||| |||||||||||||||||| ||||||||
Sbjct AGGTACTGGATGAACAGTTTACCCACCATTGAGTGGACCTATTGCTCATGGTGGATCTTC 371
Query TGNNGANNNNNCTATNNTNTCNNTNCNTNTANCCGGNTNATNNNNGATTTTAGGANNNNN 425
|| || |||| | || | | | || |||| | || ||||||||||
Sbjct TGTTGATTTAGCTATTTTTTCTTTACATTTAGCCGGTTTATCCAGGATTTTAGGAGCAAT 431
Query NNATTTTATTACTACnntttttAATATACNATCTCCNGGNATTACATTANANCNAANAAN 485
||||||||||||| |||||||||||| |||||| || ||||||||| | | || ||
Sbjct TAATTTTATTACTACAATTTTTAATATACGATCTCCAGGTATTACATTAGAACGAATAAG 491
Query ATNATTTGTATGATCNGNATTAGTTACNGCTTNTNNNCTTCTTTTATCTNTNCNNNTACT 545
|| |||||||||||| | ||||||||| |||| | |||||||||||| | | ||||
Sbjct ATTATTTGTATGATCTGTATTAGTTACAGCTTTTTTACTTCTTTTATCTTTACCAGTACT 551
Query TGCAGGGGCAATTACNANGNTTTTNACANATCGAAATTTTNNTACNACTTNTTTTGATCC 605
||||||||||||||| | | |||| ||| ||||||||||| ||| |||| |||||||||
Sbjct TGCAGGGGCAATTACAATGCTTTTAACAGATCGAAATTTTAATACCACTTTTTTTGATCC 611
Query TGCTGGAGGTGGNGATNNNATTTTATATCAACNTNNATTCTGATT 650
|||||||||||| ||| ||||||||||||| | |||||||||
Sbjct TGCTGGAGGTGGTGATCCTATTTTATATCAACATTTATTCTGATT 656
Similar but not same
Need to do phylogenetics.