The GenePool
Genomics and bioinformatics facility based within the
School of Biological Sciences, University of Edinburgh
Established 1996 with gel-based ABI373
Sequencing node of NBAF since 2001
Core funding from University of Edinburgh, SULSA, NERC
S&F
Major instrument funding from Darwin Trust, BBSRC and
NERC
MRC Next-Generation Sequencing Hub since July 2009
Technologies
2003 2008 2006 2009 2008 2009 The team
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Marian
Thomson
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Jenna
Nichols
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Nicola
Wrobel
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Alexi
Balmuth
Karolina
Grabara
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Anna
Montazam
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Jill Lovell
Denis
Cleven
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Andy
Gillies
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Urmi
Trivedi
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Timothée
Cézard
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MRC 2
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Stephen
Bridgett
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John
Davey
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MRC 3
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Anne
Wyllie
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Mark
Blaxter
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Christine
Bradbury
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Karim
Gharbi
Multiplexing using indexing
tags and adapters
Illumina multiplexing
oligonucleotide kit
Illumina multiplexing
oligonucleotide kit
Illumina multiplexing
oligonucleotide kit
Pros
Cons
Out of the box
Expensive
Supported
Alternative
workflow
Additional
sequencing
6 bases
Only 12 samples
Illumina adapters (SE)
Adapter!
5' --------------------------ACACTCTTTCCCTAC ACGACGCTCTTCCGATCT (-) -------------------- -------------- 3'!
||||||||||||||||!
3' ---------------------GTTCGTCTTCTGCCGTATGC TGCTGCGAGAAGGCTAGp (-) -------------------- -------------- 5'!
Long PCR Primer!
5' AATGATACGGCGACCACCGA GATCTACACTCTTTCCCTAC ACGACGCTCTTCCGATCT (-) -------------------- -------------- 3‘!
Short PCR Primer!
3' -------------------- -------------------- ------------------ (-) TCTAGCCTTCTCGAGCATAC GGCAGAAGACGAAC 5'!
Library fragment!
5' AATGATACGGCGACCACCGA GATCTACACTCTTTCCCTAC ACGACGCTCTTCCGATCT (N) AGATCGGAAGAGCTCGTATG CCGTCTTCTGCTTG 3'!
|||||||||||||||||||| |||||||||||||||||||| ||||||||||||||||||
|||||||||||||||||||| ||||||||||||||!
3' TTACTATGCCGCTGGTGGCT CTAGATGTGAGAAAGGGATG TGCTGCGAGAAGGCTAGA (N) TCTAGCCTTCTCGAGCATAC GGCAGAAGACGAAC 5'!
Flow cell oligo!
5’ AATGATACGGCGACCACCGA -------------------- ------------------ (N) -------------------- -------------- 3’!
Flow cell oligo!
3’ -------------------- -------------------- ------------------ (N) -------------AGCATAC GGCAGAAGACGAAC 5’!
Sequencing Primer!
5' -------------------- -----ACACTCTTTCCCTAC ACGACGCTCTTCCGATCT (-) -------------------- -------------- 3'!
Adapted from http://seqanswers.com/forums/showthread.php?t=198
Barcoded adapters
Adapter!
5' --------------------------ACACTCTTTCCCTAC ACGACGCTCTTCCGATCTxx xxxT (-) -------------------- -------------- 3'!
||||||||||||||||||| |||!
3' ---------------------GTTCGTCTTCTGCCGTATGC TGCTGCGAGAAGGCTAGAxx xxxp (-) -------------------- -------------- 5'!
Long PCR Primer!
5' AATGATACGGCGACCACCGA GATCTACACTCTTTCCCTAC ACGACGCTCTTCCGATCT-- ---- (-) -------------------- -------------- 3‘!
Short PCR Primer!
3' -------------------- -------------------- -------------------- ---- (-) ------TCTAGCCTTCTCGAGCATAC GGCAGAAGACGAAC 5'!
Library fragment!
5' AATGATACGGCGACCACCGA GATCTACACTCTTTCCCTAC ACGACGCTCTTCCGATCTxx xxxT (N) AxxxxxAGATCGGAAGAGCTCGTATG CCGTCTTCTGCTTG 3'!
|||||||||||||||||||| |||||||||||||||||||| |||||||||||||||||||| ||||
|||||||||||||||||||||||||| ||||||||||||||!
3' TTACTATGCCGCTGGTGGCT CTAGATGTGAGAAAGGGATG TGCTGCGAGAAGGCTAGAxx xxxA (N) TxxxxxTCTAGCCTTCTCGAGCATAC GGCAGAAGACGAAC 5'!
Flow cell oligo!
5’ AATGATACGGCGACCACCGA -------------------- -------------------- ---- (N) -------------------- -------------- 3’!
Flow cell oligo!
3’ -------------------- -------------------- -------------------- ---- (N) -------------AGCATAC GGCAGAAGACGAAC 5’!
Sequencing Primer!
5' -------------------- -----ACACTCTTTCCCTAC ACGACGCTCTTCCGATCT-- ---- (-) -------------------- -------------- 3'!
How to design and use
barcoded adapters
Generate MIDs of specified length and robustness with MID generator1
(may require overhang, phosphorothioate bond, 5’ phosphate and other modifications)
Check for secondary structures and illegal pairing
Append MID to adapter sequence
Order adapter from oligo supplier
Anneal matching pairs
Barcoded adapters available in the GenePool (5 bp, 3 mismatches)
64 RAD TGCA P1 adapters (28 validated)
12 standard paired-end adapters (validation in progress)
12 NlaIII tag adapters (validation in progress)
1https://www.wiki.ed.ac.uk/display/RADSequencing/Home
Still to do
Check that MIDs perform equally
Validate MID combinations
Small RNA/RNA adapters
Other indexing approaches (e.g., Epicentre transposon-based library
preparation)
Automation
Sequence capture and
targeted-resequencing
Issues to address
Which technology?
Custom design?
What coverage?
Single/paired-end?
What read length?
Multiplexing?
R&D collaborations
Species Target Size Human Exome Technology Run Coverage Indexed 34 Mb Nimblegen EZ Exome 38 Mb SureSelect All Exon 75 PE 75 PE 100X 100X No Human Exome 38 Mb SureSelect All Exon 75 PE 50X No Human 7.22p 11 Mb SureSelect 100 PE 80X Yes Mouse QTL regions 3 Mb 50 PE 60X Yes Human 22 genes (pools) 130 kb Febit HybSelect 50 PE 40X No Nimblegen custom array Still to do/investigate
Coverage of whole exome designs in the latest assembly
(GRCh37/Hg19)
Multiplexing pre- vs post-capture
Performance/optimisation of custom designs
Other sequence selection approaches (e.g., Raindance,
Fluidigm)
Automation