高通量测序技术及原理介绍.ppt

1、高通量测序技术及原理介绍,童贻刚 军事医学科学院 微生物流行病研究所 ,14,15,Illumina workflow,Sample preparation Shearing, ligate adapter Cluster generation Bridge PCR Sequencing on Genome Analyzer IIx RTA (Run Time Analysis) Analysis pipeline Offline analysis, alignment, SNPs calling, reads counting Visualize the data, reports the

2、results,Sequencing process,Fragment DNA Repair ends / Add A overhang Ligate adapters Select ligated DNA Hybridize to flow cell Extend hybridized oligos Perform bridge amplification Perform sequencing on forward strand Re-generate reverse strand,Perform sequencing on reverse strand CONFIDENTIAL DO NO

3、T DISTRIBUTE,1 Library prep ( 6 hrs) 2 Automated Cluster Generation ( 5 hrs) 1-8 samples 3 Sequencing ( 46 to 120 hrs) 1-8 samples,Sample Prep - Resequencing Surface bound adapter 1 Sequencing primer binding site Surface bound adapter 2 CONFIDENTIAL DO NOT DISTRIBUTE,CONFIDENTIAL DO NOT DISTRIBUTE,

4、Clonal clusters are generated in a contained environment (need no clean rooms) Sequencing also performed in the flow cell on the generated clusters,Flow cell 8 channels Key to the simplified workflow,Surface of flow cell coated with a lawn of oligo pairs,Cluster generation: Hybridize fragment & exte

5、nd Adapter sequence 50 M single molecules hybridize to the lawn of primers Bound molecules are then extended by polymerases 3 extension CONFIDENTIAL DO NOT DISTRIBUTE,Double-stranded,molecule is denatured. Original template is washed away.,Newly synthesized covalently attached to the flow cell surfa

6、ce. CONFIDENTIAL DO NOT DISTRIBUTE,Cluster generation: Denature double-stranded DNA Newly,synthesized,strand,Original template discard,Cluster generation: Covalently bound spatially separated single molecules Single molecules bound to flow cell in a random pattern CONFIDENTIAL DO NOT DISTRIBUTE,Clus

7、ter generation: Bridge amplification Single-strand flips over to hybridize to adjacent primers to form a bridge. Hybridized primer is extended by polymerases. CONFIDENTIAL DO NOT DISTRIBUTE,Cluster generation: Bridge amplification double-stranded bridge is formed. CONFIDENTIAL DO NOT DISTRIBUTE,Clus

8、ter generation: Bridge amplification Double-stranded bridge is denatured. Result: Two copies of covalently bound single- stranded templates. CONFIDENTIAL DO NOT DISTRIBUTE,Cluster generation: Bridge amplification Single-strands flip over to hybridize to adjacent primers to form bridges. Hybridized p

9、rimer is extended by polymerase. CONFIDENTIAL DO NOT DISTRIBUTE,Cluster generation: Bridge amplification Bridge amplification cycle repeated till multiple bridges are formed CONFIDENTIAL DO NOT DISTRIBUTE,Cluster generation dsDNA bridges denatured. Reverse strands cleaved and washed away. CONFIDENTI

10、AL DO NOT DISTRIBUTE,Cluster generation leaving a cluster with forward strands only. CONFIDENTIAL DO NOT DISTRIBUTE,Cluster generation Free 3 ends are blocked to prevent unwanted DNA priming. CONFIDENTIAL DO NOT DISTRIBUTE,CONFIDENTIAL DO NOT DISTRIBUTE,hybridized to adapter sequence.,Sequencing Seq

11、uencing primer is,Sequencing primer,Add 4 Fl- NTPs + Polymerase,Incorporated Fl-NTP is imaged,Terminator and fluorescent dye are cleaved from the Fl-NTP,X 36 CONFIDENTIAL DO NOT DISTRIBUTE,Sequencing primer,Flow cell imaging Total Internal Reflection Fluorescence Fluidics port Flow cell Prism Fluidi

12、cs port CONFIDENTIAL DO NOT DISTRIBUTE,CONFIDENTIAL DO NOT DISTRIBUTE,Paired end sequencing Sequenced,strand stripped off,3-ends unblocked,Paired end sequencing Bridge formation 3 extension CONFIDENTIAL DO NOT DISTRIBUTE,Paired end sequencing Double stranded DNA is denatured CONFIDENTIAL DO NOT DIST

13、RIBUTE,Paired end sequencing 3 ends are blocked Original forward strand is cleaved CONFIDENTIAL DO NOT DISTRIBUTE,Add 4 Fl- NTPs + Polymerase,Incorporated Fl-NTP is imaged,Terminator and fluorescent dye are cleaved from the Fl-NTP,X 36 - 50 CONFIDENTIAL DO NOT DISTRIBUTE,Sequencing reverse strand Hy

14、bridize sequencing primer,Solexa,Flow cell in GAIIx,CONFIDENTIAL DO NOT DISTRIBUTE,Image re-analysis pipleline,Image Analysis Base calling Sequence Analysis,GA Analysis Pipeline,Instrument PC,Analysis PC/cluster,data transfer,Images (.tif) Lane 1.8 Cycle 1.36 Tile_Cycle_Image_a, Tile_Cycle_Image_c,

15、Tile_Cycle_Image_g, Tile_Cycle_Image_t .params file,For each tile: Cluster intensities Cluster noise For each tile: Corrected cluster intensities Cluster sequence Cluster probabilities For all data: Quality Filtering Sequence Alignment Run Statistics Visualization,CONFIDENTIAL DO NOT DISTRIBUTE,Bust

16、ard,Base with highest corrected intensity is called,A,C,G,T,C,Gerald,I A I A + IB,GEneration of,Recursive Analyses Linked by,Dependency,IA IB,Filtering removes low quality base calls Chastity: C =,Default value 0.6 Other filters include purity, similarity, neighbor and neighborhood. CONFIDENTIAL DO

17、NOT DISTRIBUTE,Bustard output *_qseq.txt,Machine name,Run number,Lane number,Tile number,X coord,Y coord,Sequence,Quality,PassedFilter,Index,Read format,EAS1 89 1 59 111 525 AACCTT 2 TGACCAGCGTCAACCAGTACTACGTCTTTGTCGATAG aaaaa_V_OYOZZYUPJZRX 1 EAS1 89 1 59 111 726 AACCTT 2 TCTGGATGAAGAACGATCCGCTGCAG

18、AGGTGCTGGCA _FNXXZWFZ_YYTYMUVBBBBBBBBBBB 0 EAS1 89 1 59 111 860 AACCTT 2 TATCGCGTAGTGTAGCACGGCCTTTTTTTCGTCCACC aaaXFUWQUHVN_ZRWZZXFWYFTX 1 EAS1 89 1 59 112 377 AACCTT 2 TTTTCTTCTCCTTCGCCATCAGCGACAAAATCAAGCA abbbabbbbbbaaaTaaaaaY_YNaZZ 1 EAS1 89 1 59 112 538 AACCTT 2 TGTGAATTAACAGTATTGGCGTAGTTACAGGCAGTGT aa_aabbaaa_aSYZYUBBBBB 1 EAS1 89 1 59 112 576 AACCTT 2 TCTCCTTCGTCTTCTTCCATCAGTTGTTCGACCGGCT GJRNGBBBBBBBBBBBBBBBBBBBBBBBBBBBBB 0 EAS1 89 1 59 112 607 AACCTT 2 TCCACCATCAACTGGTTGCCAGTGCGCGGGCAGTTAA aabaaaaaaX_YTTHTTZQYTX 1 EAS1 89 1 59 112 255 AACCTT 2 TGATGCTGATAAGCAGCGTGCTCACAACCCAGATTTG