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Bng presentation draft

Jennifer Shelton, profile picture
Jennifer Shelton

The document discusses algorithms and filters utilized to enhance the tribolium draft assembly through the integration of physical maps based on ultra-long single DNA molecules. It presents the pipeline's results, indicating the estimated genome size, alignment coverage, and the effectiveness of using bng assembled molecules which yielded higher quality scaffolding. The findings emphasize the challenges faced during assembly, including potential misalignments and redundancy in contigs.

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Algorithms and filters used to improve the Tribolium draft Assembly with Physical Maps Based on Imaging Ultra-Long Single DNA Molecules ! Jennifer Shelton 2014
Assembly Pipeline 3) use sequence reference to adjust molecule stretch for each scan
Assembly Pipeline In recent datasets when SNR is low and alignment is good we see a spike in bases per pixel (bpp) in the first scan, a plateau and a lower plateau First scan in a flow cell
Assembly Pipeline 5) Use sequence reference to determine assembly noise parameters. Estimated genome size is used to set the p-value threshold.
Assembly Pipeline 6/7) Variants of the starting p-value and default minimum molecule length are explored in nine assemblies.
Current Tribolium sequence-based assembly Input file N50 (Mb) Number of Contigs Cumulative Length (Mb) Genome FASTA 1.16 2240 160.74 in silico CMAP from FASTA 1.20 223 152.53 223 scaffolds from the sequence-based assembly were longer than 20 (kb) with more than 5 labels and were converted into in silico CMAPs
Assembly Results Input file N50 (Mb) Number of Contigs Cumulative Length (Mb) Genome FASTA 1.16 2240 160.74 in silico CMAP from FASTA 1.20 223 152.53 CMAP from assembled BNG molecules (BNG CMAP) 1.35 216 200.47 BNG assembled molecules had a higher N50 and longer cumulative length than the sequence assembly ! The estimated size of the Tribolium genome is ~200 (Mb)
Simplest XMAP alignment description 1 (Mb) 1.1 (Mb) 1.1 (Mb) 1.3 (Mb) Breadth of alignment coverage for in silico CMAP: 2.1 (Mb) Total alignment length for in silico CMAP: 2.1 (Mb) ! Breadth of alignment coverage for BNG CMAP: 2.4 (Mb) Total alignment length for BNG CMAP: 2.4 (Mb) in silico CMAP from genome FASTA CMAP from assembled molecules in silico CMAP 1 in silico CMAP 2 BNG CMAP 1 BNG CMAP 2
Complex XMAP alignment description 1 (Mb) in silico CMAP 1 BNG CMAP 1 BNG CMAP 2 1.1 (Mb) 1.3 (Mb) Breadth of alignment coverage for in silico CMAP: 1 (Mb) Total alignment length for in silico CMAP: 2 (Mb) ! Breadth of alignment coverage for BNG CMAP: 2.4 (Mb) Total alignment length for BNG CMAP: 2.4 (Mb) in silico CMAP from genome FASTA CMAP from assembled molecules
Alignment of CMAPs 1 (Mb) in silico CMAP 1 BNG CMAP 1 BNG CMAP 2 1.1 (Mb) 1.3 (Mb) Breadth of alignment coverage compared to total aligned length can indicate relevant relationships between assemblies ! In this example differences between "breadth" and "total" length could be due to: ! Duplications in sample molecules were extracted from Assembly of alternate haplotypes Mis-assembly creating redundant contigs Collapsed repeat in sequence assembly in silico CMAP from genome FASTA CMAP from assembled molecules
Alignment of BNG assembly to reference genome CMAP name Breadth of alignment coverage for CMAP (Mb) Length of total alignment for CMAP (Mb) Percent of CMAP aligned in silico CMAP from FASTA 124.04 132.40 81 CMAP from assembled BNG molecules (BNG CMAP) 131.64 132.34 67 Close to 4% of the alignment of the in silico CMAP appears to be redundant ! Overall 81% of the in silico CMAP aligns to the BNG consensus map
ChLG 9 super! Alignment of BNG assembly to reference genome scaffold BNG consensus maps ChLG 9! scaffolds 130 131 133 134 132 129 135 127 136 137 BNG consensus Typically where redundant alignments occur two BNG consensus maps aligned suggesting they represent haplotypes although this has not been verified maps
Tribolium super-scaffolds overlapping BNG cmap ChLG 9 super! scaffold BNG consensus maps ChLG 9! scaffolds 128 130 131 133 134 132 BNG consensus maps
Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds + in silico CMAP 1 + in silico CMAP 4 Stitch.pl estimates super scaffolds using alignments of scaffolds and assembled BNG molecules using BNG Refaligner in silico CMAP aligned as reference + in silico CMAP 2 - in silico CMAP 3 BNG CMAP 1 BNG CMAP 2
Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds + in silico CMAP 1 + in silico CMAP 4 BNG CMAP 1 BNG CMAP 2 + in silico CMAP 1 + in silico CMAP 4 Stitch.pl estimates super scaffolds using alignments of scaffolds and assembled BNG molecules using BNG Refaligner in silico CMAP aligned as reference alignment is inverted and used as input for stitch + in silico CMAP 2 - in silico CMAP 3 BNG CMAP 1 BNG CMAP 2 + in silico CMAP 2 - in silico CMAP 3
Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds + in silico CMAP 1 + in silico CMAP 4 BNG CMAP 1 BNG CMAP 2 + in silico CMAP 1 + in silico CMAP 4 BNG CMAP 1 BNG CMAP 2 Stitch.pl estimates super scaffolds using alignments of scaffolds and assembled BNG molecules using BNG Refaligner in silico CMAP aligned as reference alignment is inverted and used as input for stitch + in silico CMAP 2 - in silico CMAP 3 + in silico CMAP 4 alignments are filtered based on alignment length relative total possible alignment length and confidence + in silico CMAP 2 - in silico CMAP 3 BNG CMAP 1 BNG CMAP 2 + in silico CMAP 2 - in silico CMAP 3 + in silico CMAP 1
Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds BNG CMAP 1 BNG CMAP 2 + in silico CMAP 1 + in silico CMAP 4 + in silico CMAP 2 - in silico CMAP 3 BNG CMAP 1 + in silico CMAP 1 Stitch.pl checks alignment length against potential alignment lengths to find relevant global rather than local alignments alignment passes because the alignment length is greater than 30% of the potential alignment length
Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium BNG CMAP 1 BNG CMAP 2 + in silico CMAP 1 + in silico CMAP 4 + in silico CMAP 2 - in silico CMAP 3 BNG CMAP 1 scaffolds + in silico CMAP 2 Stitch.pl checks alignment length against potential alignment lengths to find relevant global rather than local alignments alignment passes because the alignment length is greater than 30% of the potential alignment length
Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds BNG CMAP 1 BNG CMAP 2 + in silico CMAP 1 + in silico CMAP 4 + in silico CMAP 2 - in silico CMAP 3 - in silico CMAP 2 BNG CMAP 2 Stitch.pl checks alignment length against potential alignment lengths to find relevant global rather than local alignments alignment passes because the alignment length is greater than 30% of the potential alignment length
Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds BNG CMAP 1 BNG CMAP 2 + in silico CMAP 1 + in silico CMAP 4 + in silico CMAP 2 - in silico CMAP 3 - in silico CMAP 2 BNG CMAP 2 Stitch.pl checks alignment length against potential alignment lengths to find relevant global rather than local alignments alignment fails because the alignment length is less than 30% of the potential alignment length
Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds BNG CMAP 1 BNG CMAP 2 + in silico CMAP 1 + in silico CMAP 4 + in silico CMAP 2 - in silico CMAP 3 + in silico CMAP 2 BNG CMAP 2 Stitch.pl checks alignment length against potential alignment lengths to find relevant global rather than local alignments alignment fails because the alignment length is less than 30% of the potential alignment length
Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds BNG CMAP 1 BNG CMAP 2 + in silico CMAP 1 + in silico CMAP 4 + in silico CMAP 2 - in silico CMAP 3 BNG CMAP 2 Stitch.pl checks alignment length against potential alignment lengths to find relevant global rather than local alignments alignment passes because the alignment length is greater than 30% of the potential alignment length - in silico CMAP 3
Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium BNG CMAP 1 BNG CMAP 2 + in silico CMAP 1 + in silico CMAP 4 + in silico CMAP 2 - in silico CMAP 3 BNG CMAP 2 scaffolds Stitch.pl checks alignment length against potential alignment lengths to find relevant global rather than local alignments alignment fails because the alignment length is less than 30% of the potential alignment length - in silico CMAP 3
Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium BNG CMAP 1 BNG CMAP 2 + in silico CMAP 1 + in silico CMAP 4 + in silico CMAP 2 - in silico CMAP 3 BNG CMAP 2 scaffolds Stitch.pl checks alignment length against potential alignment lengths to find relevant global rather than local alignments alignment passes because the alignment length is greater than 30% of the potential alignment length + in silico CMAP 4
Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds BNG CMAP 1 BNG CMAP 2 + in silico CMAP 2 - in silico CMAP 3 + in silico CMAP 4 high quality scaffolding alignments... + in silico CMAP 1
Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds are filtered for longest and highest confidence alignment for each in silico CMAP BNG CMAP 1 BNG CMAP 2 + in silico CMAP 2 - in silico CMAP 3 BNG CMAP 1 BNG CMAP 2 + in silico CMAP 2 - in silico CMAP 3 + in silico CMAP 4 + in silico CMAP 1 + in silico CMAP 4 high quality scaffolding alignments... + in silico CMAP 1
Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds are filtered for longest and highest confidence alignment for each in silico CMAP Passing alignments are used to super scaffold BNG CMAP 1 BNG CMAP 2 + in silico CMAP 2 - in silico CMAP 3 BNG CMAP 1 BNG CMAP 2 + in silico CMAP 2 - in silico CMAP 3 + in silico CMAP 4 + in silico CMAP 1 + in silico CMAP 4 BNG CMAP 1 BNG CMAP 2 + in silico CMAP 2 - in silico CMAP 3 + in silico CMAP 1 + in silico CMAP 4 high quality scaffolding alignments... + in silico CMAP 1
Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds Stitch is iterated and additional super scaffolding alignments are found BNG CMAP 1 BNG CMAP 2 + in silico CMAP 2 - in silico CMAP 3 + in silico CMAP 1 + in silico CMAP 4 Iteration takes advantage of alignments where sequence-based scaffolds stitch BNG consensus maps
Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds Stitch is iterated and additional super scaffolding alignments are found BNG CMAP 1 BNG CMAP 2 + in silico CMAP 2 - in silico CMAP 3 + in silico CMAP 1 + in silico CMAP 4 Until all super scaffolds are BNG CMAP 1 BNG CMAP 2 joined + in silico CMAP 2 - in silico CMAP 3 + in silico CMAP 1 + in silico CMAP 4 Iteration takes advantage of alignments where sequence-based scaffolds stitch BNG consensus maps
Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds BNG CMAP 1 BNG CMAP 2 - in silico CMAP 3 + in silico CMAP 2 + in silico CMAP 4 + in silico CMAP 1 If gap length is estimated to be negative gaps are represented by 100 (bp) fillers
Gap lengths Distribution of gap lengths for automated output Gap length (bp) Of the automated stitch.pl Tribolium super-scaffolds there were 66 gaps had known lengths and 26 had negative lengths (set to 100 (bp)) ! Of the manually edited Tribolium super-scaffolds there were 66 gaps had known lengths and 24 had negative lengths (set to 100 (bp)) Count −1500000 −1000000 −500000 0 500000 1000000 0 5 10 15 20 Negative gap lengths Positive gap lengths
Gap lengths Distribution of gap lengths for automated output Gap length (bp) Of the automated stitch.pl Tribolium super-scaffolds there were 66 gaps had known lengths and 26 had negative lengths (set to 100 (bp)) ! Of the manually edited Tribolium super-scaffolds there were 66 gaps had known lengths and 24 had negative lengths (set to 100 (bp)) Count −1500000 −1000000 −500000 0 500000 1000000 0 5 10 15 20 Negative gap lengths Positive gap lengths
Negative gap lengths Is part of scaffold_23 connected to 136?! I went with the second alignment (21-26 together and 136-137 together because it is supported by genetic maps) but we should check these assemblies. ! ! In bottom alignment of 136 you can see that a large section of the BNG map 32 (which joins 23 to 136) is a duplicate in the BNG assembly? 22 23 129 136 137 The longest negative gap length is from a BNG consenus map joining in silico 23 and 136
Negative gap lengths Is part of scaffold_23 connected to 136?! I went with the second alignment (21-26 together and 136-137 together because it is supported by genetic maps) but we should check these assemblies. ! ! In bottom alignment of 136 you can see that a large section of the BNG map 32 (which joins 23 to 136) is a duplicate in the BNG assembly? 22 23 129 136 137 ! Because the same region of 136 aligns to another BNG consensus map that aligns to its chromosome linkage group this alignment was rejected and stitch was re-run
scaffold_133 aligns but is not visible in IrysView?) why is Super_scaffold_65 backwards? Negative gap lengths ChLG 2 super! scaffold ChLG 2 super! scaffold BNG consensus maps BNG consensus maps ChLG 2! scaffolds 133 134 132 129 135 127 136 137 139 138 140 141 142 143 144 145 Two new super scaffolds were created and the sequence similarity is being evaluated min confidence 10 scaffold_133 aligns but is not visible in IrysView?) why is Super_scaffold_65 backwards? ChLG 2! scaffolds 130 131 133 134 132 129 135 127 136 137 139 138 140 141 142 143 144 145 U 18 14 16 19 20 21 22 23 24 25 26 27 28 30 BNG consensus maps U 18 14 16 19 20 21 22 23 24 25 26 27 28 30 BNG consensus maps
Gap lengths Distribution of gap lengths for automated output Gap length (bp) This negative alignment also indicated a potential assembly issue Count −1500000 −1000000 −500000 0 500000 1000000 0 5 10 15 20 Negative gap lengths Positive gap lengths
Negative gap lengths This negative gap length is from a BNG consenus map joining in silico 81 and 102 and 103 Half of scaffold_81 aligns with ChLG7
Negative gap lengths Half of scaffold_81 aligns with ChLG7 79 80 81 82 83 Because the other half of 81 aligns to another BNG consensus map that aligns to its chromosome linkage group this alignment was rejected and stitch was re-run ! The BNG maps suggest a mis-assembly of in silico 81 at a sequence level
Distribution of gap lengths for automated output Gap length (bp) Count −1500000 −1000000 −500000 0 500000 1000000 0 5 10 15 20 Negative gap lengths Positive gap lengths Gap lengths All extremely small negative gap lengths, < -40,000 (bp) (shaded), were independently flagged as potential sequence mis-assemblies to be checked at the sequence-level
Distribution of gap lengths for automated output Gap length (bp) Count −1500000 −1000000 −500000 0 500000 1000000 0 5 10 15 20 Negative gap lengths Positive gap lengths Gap lengths All gaps from the shaded regions were also manually rejected and stitch.pl was rerun without them for the current super-scaffolded assembly ! We suspect extremely small negative gap sizes may be useful in locating sequence mis-assemblies
Tribolium super-scaffolds Input file N50 (Mb) Number of Contigs Cumulative Length (Mb) genome FASTA 1.16 2240 160.74 super-scaffold FASTA 4.46 2150 165.92 N50 of the super-scaffolded genome was ~4 times greater than the original ! Super-scaffolds tend to agree with the Tribolium genetic map
Tribolium super-scaffolds Input file N50 (Mb) Number of Contigs genome FASTA 1.16 2240 160.74 4.46 2150 165.92 For Tribolium : first minimum percent aligned = 30% first minimum confidence = 13 Cumulative Length (Mb) second minimum percent aligned = 90% second minimum confidence = 8 ! super-scaffold FASTA Lower quality alignments were manually selected if genetic map also supported the order Complex scaffolds were broken manually for sequence level evaluation
Tribolium super-scaffolds min confidence 10 From ChLGX, 11 of the previous 13 scaffolds were joined with two unplaced scaffolds (U) into one super scaffold. ChLG X was reduced from 13 scaffolds to 2 with one scaffold being moved to ChLG 3 ChLG X super! scaffold BNG consensus maps ChLG X! scaffolds BNG consensus maps U 3 4 5 6 7 U 8 9 10 11 12 13
Tribolium super-scaffolds min confidence 10 51 U 43 45 44 46 The second scaffold from ChLG X aligned to scaffolds from a portion of ChLG 3 ChLG 3 super! scaffold BNG consensus maps ChLG 3! scaffolds BNG consensus maps 32 33 34 35 36 2 37 38 39 40 41 42 ChLG 3 super! scaffold BNG consensus maps ChLG 3 super! scaffold BNG consensus
Tribolium super-scaffolds min confidence 10 From ChLGX, 11 of the previous 13 scaffolds were joined with two unplaced scaffolds (U) into one super scaffold. Two unplaced scaffolds aligned to ChLG X ChLG X super! scaffold BNG consensus maps ChLG X! scaffolds BNG consensus maps U 3 4 5 6 7 U 8 9 10 11 12 13
Tribolium super-scaffolds min confidence 10 From ChLGX, 11 of the previous 13 scaffolds were joined with two unplaced scaffolds (U) into one super scaffold. 4% Redundancy in alignment may be from assembly of haplotypes (generally observed as two BNG consensus maps aligning to the same in silico map) ChLG X super! scaffold BNG consensus maps ChLG X! scaffolds BNG consensus maps U 3 4 5 6 7 U 8 9 10 11 12 13
Tribolium super-scaffolds overlapping BNG cmap min confidence 10 From ChLGX, 11 of the previous 13 scaffolds were joined with two unplaced scaffolds (U) into one super scaffold. ChLG X super! scaffold BNG consensus maps ChLG X! scaffolds BNG consensus maps U 3 4 5 6 7 U 8 9 10 11 12 13
Tribmoinl icuonmfid esnucep 1e0r-scaffolds ChLG9 currently (2150 scaffold_133 aligns but is not visible in IrysView?) why is Super_scaffold_65 backwards? 128 130 131 133 134 132 129 135 127 136 137 139 138 140 141 142 143 144 145 For ChLG 9 21 scaffolds were reduced to 9 ChLG 9 super! scaffold BNG consensus maps ChLG 9! scaffolds BNG consensus maps
min confidence 10 Tribolium super-scaffolds For ChLG 5 17 scaffolds were reduced to 4 ChLG 5 super! scaffold BNG consensus maps ChLG 5! scaffolds BNG consensus maps 69 68 70 71 72 73 74 U 75 76 77 78 79 80 81 82 83
Acknowledgements K-INBRE Bioinformatics Core! Susan Brown - PI Nic Herndon - script development Nanyan Lu - manual editing Michelle Coleman - extractions and running the Irys! Zachary Sliefert - metric summaries ! Bionano Genomics! Ernest Lam - assembly pipeline best practices assistance Weiping Wang - assistance with data formats Palak Sheth - collaboration to standardize analysis ! Script availability! https://github.com/i5K-KINBRE-script-share/Irys-scaffolding BNG scripts available by request from BNG
Gap lengths Distribution of gap lengths for automated output Gap length (bp) Of the automated stitch.pl Tribolium super-scaffolds there were 66 gaps had known lengths and 26 had negative lengths (set to 100 (bp)) ! Of the manually edited Tribolium super-scaffolds there were 66 gaps had known lengths and 24 had negative lengths (set to 100 (bp)) Count −1500000 −1000000 −500000 0 500000 1000000 0 5 10 15 20 Negative gap lengths Positive gap lengths

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Bng presentation draft

  • 1. Algorithms and filters used to improve the Tribolium draft Assembly with Physical Maps Based on Imaging Ultra-Long Single DNA Molecules ! Jennifer Shelton 2014
  • 2. Assembly Pipeline 3) use sequence reference to adjust molecule stretch for each scan
  • 3. Assembly Pipeline In recent datasets when SNR is low and alignment is good we see a spike in bases per pixel (bpp) in the first scan, a plateau and a lower plateau First scan in a flow cell
  • 4. Assembly Pipeline 5) Use sequence reference to determine assembly noise parameters. Estimated genome size is used to set the p-value threshold.
  • 5. Assembly Pipeline 6/7) Variants of the starting p-value and default minimum molecule length are explored in nine assemblies.
  • 6. Current Tribolium sequence-based assembly Input file N50 (Mb) Number of Contigs Cumulative Length (Mb) Genome FASTA 1.16 2240 160.74 in silico CMAP from FASTA 1.20 223 152.53 223 scaffolds from the sequence-based assembly were longer than 20 (kb) with more than 5 labels and were converted into in silico CMAPs
  • 7. Assembly Results Input file N50 (Mb) Number of Contigs Cumulative Length (Mb) Genome FASTA 1.16 2240 160.74 in silico CMAP from FASTA 1.20 223 152.53 CMAP from assembled BNG molecules (BNG CMAP) 1.35 216 200.47 BNG assembled molecules had a higher N50 and longer cumulative length than the sequence assembly ! The estimated size of the Tribolium genome is ~200 (Mb)
  • 8. Simplest XMAP alignment description 1 (Mb) 1.1 (Mb) 1.1 (Mb) 1.3 (Mb) Breadth of alignment coverage for in silico CMAP: 2.1 (Mb) Total alignment length for in silico CMAP: 2.1 (Mb) ! Breadth of alignment coverage for BNG CMAP: 2.4 (Mb) Total alignment length for BNG CMAP: 2.4 (Mb) in silico CMAP from genome FASTA CMAP from assembled molecules in silico CMAP 1 in silico CMAP 2 BNG CMAP 1 BNG CMAP 2
  • 9. Complex XMAP alignment description 1 (Mb) in silico CMAP 1 BNG CMAP 1 BNG CMAP 2 1.1 (Mb) 1.3 (Mb) Breadth of alignment coverage for in silico CMAP: 1 (Mb) Total alignment length for in silico CMAP: 2 (Mb) ! Breadth of alignment coverage for BNG CMAP: 2.4 (Mb) Total alignment length for BNG CMAP: 2.4 (Mb) in silico CMAP from genome FASTA CMAP from assembled molecules
  • 10. Alignment of CMAPs 1 (Mb) in silico CMAP 1 BNG CMAP 1 BNG CMAP 2 1.1 (Mb) 1.3 (Mb) Breadth of alignment coverage compared to total aligned length can indicate relevant relationships between assemblies ! In this example differences between "breadth" and "total" length could be due to: ! Duplications in sample molecules were extracted from Assembly of alternate haplotypes Mis-assembly creating redundant contigs Collapsed repeat in sequence assembly in silico CMAP from genome FASTA CMAP from assembled molecules
  • 11. Alignment of BNG assembly to reference genome CMAP name Breadth of alignment coverage for CMAP (Mb) Length of total alignment for CMAP (Mb) Percent of CMAP aligned in silico CMAP from FASTA 124.04 132.40 81 CMAP from assembled BNG molecules (BNG CMAP) 131.64 132.34 67 Close to 4% of the alignment of the in silico CMAP appears to be redundant ! Overall 81% of the in silico CMAP aligns to the BNG consensus map
  • 12. ChLG 9 super! Alignment of BNG assembly to reference genome scaffold BNG consensus maps ChLG 9! scaffolds 130 131 133 134 132 129 135 127 136 137 BNG consensus Typically where redundant alignments occur two BNG consensus maps aligned suggesting they represent haplotypes although this has not been verified maps
  • 13. Tribolium super-scaffolds overlapping BNG cmap ChLG 9 super! scaffold BNG consensus maps ChLG 9! scaffolds 128 130 131 133 134 132 BNG consensus maps
  • 14. Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds + in silico CMAP 1 + in silico CMAP 4 Stitch.pl estimates super scaffolds using alignments of scaffolds and assembled BNG molecules using BNG Refaligner in silico CMAP aligned as reference + in silico CMAP 2 - in silico CMAP 3 BNG CMAP 1 BNG CMAP 2
  • 15. Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds + in silico CMAP 1 + in silico CMAP 4 BNG CMAP 1 BNG CMAP 2 + in silico CMAP 1 + in silico CMAP 4 Stitch.pl estimates super scaffolds using alignments of scaffolds and assembled BNG molecules using BNG Refaligner in silico CMAP aligned as reference alignment is inverted and used as input for stitch + in silico CMAP 2 - in silico CMAP 3 BNG CMAP 1 BNG CMAP 2 + in silico CMAP 2 - in silico CMAP 3
  • 16. Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds + in silico CMAP 1 + in silico CMAP 4 BNG CMAP 1 BNG CMAP 2 + in silico CMAP 1 + in silico CMAP 4 BNG CMAP 1 BNG CMAP 2 Stitch.pl estimates super scaffolds using alignments of scaffolds and assembled BNG molecules using BNG Refaligner in silico CMAP aligned as reference alignment is inverted and used as input for stitch + in silico CMAP 2 - in silico CMAP 3 + in silico CMAP 4 alignments are filtered based on alignment length relative total possible alignment length and confidence + in silico CMAP 2 - in silico CMAP 3 BNG CMAP 1 BNG CMAP 2 + in silico CMAP 2 - in silico CMAP 3 + in silico CMAP 1
  • 17. Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds BNG CMAP 1 BNG CMAP 2 + in silico CMAP 1 + in silico CMAP 4 + in silico CMAP 2 - in silico CMAP 3 BNG CMAP 1 + in silico CMAP 1 Stitch.pl checks alignment length against potential alignment lengths to find relevant global rather than local alignments alignment passes because the alignment length is greater than 30% of the potential alignment length
  • 18. Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium BNG CMAP 1 BNG CMAP 2 + in silico CMAP 1 + in silico CMAP 4 + in silico CMAP 2 - in silico CMAP 3 BNG CMAP 1 scaffolds + in silico CMAP 2 Stitch.pl checks alignment length against potential alignment lengths to find relevant global rather than local alignments alignment passes because the alignment length is greater than 30% of the potential alignment length
  • 19. Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds BNG CMAP 1 BNG CMAP 2 + in silico CMAP 1 + in silico CMAP 4 + in silico CMAP 2 - in silico CMAP 3 - in silico CMAP 2 BNG CMAP 2 Stitch.pl checks alignment length against potential alignment lengths to find relevant global rather than local alignments alignment passes because the alignment length is greater than 30% of the potential alignment length
  • 20. Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds BNG CMAP 1 BNG CMAP 2 + in silico CMAP 1 + in silico CMAP 4 + in silico CMAP 2 - in silico CMAP 3 - in silico CMAP 2 BNG CMAP 2 Stitch.pl checks alignment length against potential alignment lengths to find relevant global rather than local alignments alignment fails because the alignment length is less than 30% of the potential alignment length
  • 21. Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds BNG CMAP 1 BNG CMAP 2 + in silico CMAP 1 + in silico CMAP 4 + in silico CMAP 2 - in silico CMAP 3 + in silico CMAP 2 BNG CMAP 2 Stitch.pl checks alignment length against potential alignment lengths to find relevant global rather than local alignments alignment fails because the alignment length is less than 30% of the potential alignment length
  • 22. Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds BNG CMAP 1 BNG CMAP 2 + in silico CMAP 1 + in silico CMAP 4 + in silico CMAP 2 - in silico CMAP 3 BNG CMAP 2 Stitch.pl checks alignment length against potential alignment lengths to find relevant global rather than local alignments alignment passes because the alignment length is greater than 30% of the potential alignment length - in silico CMAP 3
  • 23. Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium BNG CMAP 1 BNG CMAP 2 + in silico CMAP 1 + in silico CMAP 4 + in silico CMAP 2 - in silico CMAP 3 BNG CMAP 2 scaffolds Stitch.pl checks alignment length against potential alignment lengths to find relevant global rather than local alignments alignment fails because the alignment length is less than 30% of the potential alignment length - in silico CMAP 3
  • 24. Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium BNG CMAP 1 BNG CMAP 2 + in silico CMAP 1 + in silico CMAP 4 + in silico CMAP 2 - in silico CMAP 3 BNG CMAP 2 scaffolds Stitch.pl checks alignment length against potential alignment lengths to find relevant global rather than local alignments alignment passes because the alignment length is greater than 30% of the potential alignment length + in silico CMAP 4
  • 25. Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds BNG CMAP 1 BNG CMAP 2 + in silico CMAP 2 - in silico CMAP 3 + in silico CMAP 4 high quality scaffolding alignments... + in silico CMAP 1
  • 26. Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds are filtered for longest and highest confidence alignment for each in silico CMAP BNG CMAP 1 BNG CMAP 2 + in silico CMAP 2 - in silico CMAP 3 BNG CMAP 1 BNG CMAP 2 + in silico CMAP 2 - in silico CMAP 3 + in silico CMAP 4 + in silico CMAP 1 + in silico CMAP 4 high quality scaffolding alignments... + in silico CMAP 1
  • 27. Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds are filtered for longest and highest confidence alignment for each in silico CMAP Passing alignments are used to super scaffold BNG CMAP 1 BNG CMAP 2 + in silico CMAP 2 - in silico CMAP 3 BNG CMAP 1 BNG CMAP 2 + in silico CMAP 2 - in silico CMAP 3 + in silico CMAP 4 + in silico CMAP 1 + in silico CMAP 4 BNG CMAP 1 BNG CMAP 2 + in silico CMAP 2 - in silico CMAP 3 + in silico CMAP 1 + in silico CMAP 4 high quality scaffolding alignments... + in silico CMAP 1
  • 28. Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds Stitch is iterated and additional super scaffolding alignments are found BNG CMAP 1 BNG CMAP 2 + in silico CMAP 2 - in silico CMAP 3 + in silico CMAP 1 + in silico CMAP 4 Iteration takes advantage of alignments where sequence-based scaffolds stitch BNG consensus maps
  • 29. Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds Stitch is iterated and additional super scaffolding alignments are found BNG CMAP 1 BNG CMAP 2 + in silico CMAP 2 - in silico CMAP 3 + in silico CMAP 1 + in silico CMAP 4 Until all super scaffolds are BNG CMAP 1 BNG CMAP 2 joined + in silico CMAP 2 - in silico CMAP 3 + in silico CMAP 1 + in silico CMAP 4 Iteration takes advantage of alignments where sequence-based scaffolds stitch BNG consensus maps
  • 30. Alignment of BNG assembly to reference genome was used to super-scaffold the Tribolium scaffolds BNG CMAP 1 BNG CMAP 2 - in silico CMAP 3 + in silico CMAP 2 + in silico CMAP 4 + in silico CMAP 1 If gap length is estimated to be negative gaps are represented by 100 (bp) fillers
  • 31. Gap lengths Distribution of gap lengths for automated output Gap length (bp) Of the automated stitch.pl Tribolium super-scaffolds there were 66 gaps had known lengths and 26 had negative lengths (set to 100 (bp)) ! Of the manually edited Tribolium super-scaffolds there were 66 gaps had known lengths and 24 had negative lengths (set to 100 (bp)) Count −1500000 −1000000 −500000 0 500000 1000000 0 5 10 15 20 Negative gap lengths Positive gap lengths
  • 32. Gap lengths Distribution of gap lengths for automated output Gap length (bp) Of the automated stitch.pl Tribolium super-scaffolds there were 66 gaps had known lengths and 26 had negative lengths (set to 100 (bp)) ! Of the manually edited Tribolium super-scaffolds there were 66 gaps had known lengths and 24 had negative lengths (set to 100 (bp)) Count −1500000 −1000000 −500000 0 500000 1000000 0 5 10 15 20 Negative gap lengths Positive gap lengths
  • 33. Negative gap lengths Is part of scaffold_23 connected to 136?! I went with the second alignment (21-26 together and 136-137 together because it is supported by genetic maps) but we should check these assemblies. ! ! In bottom alignment of 136 you can see that a large section of the BNG map 32 (which joins 23 to 136) is a duplicate in the BNG assembly? 22 23 129 136 137 The longest negative gap length is from a BNG consenus map joining in silico 23 and 136
  • 34. Negative gap lengths Is part of scaffold_23 connected to 136?! I went with the second alignment (21-26 together and 136-137 together because it is supported by genetic maps) but we should check these assemblies. ! ! In bottom alignment of 136 you can see that a large section of the BNG map 32 (which joins 23 to 136) is a duplicate in the BNG assembly? 22 23 129 136 137 ! Because the same region of 136 aligns to another BNG consensus map that aligns to its chromosome linkage group this alignment was rejected and stitch was re-run
  • 35. scaffold_133 aligns but is not visible in IrysView?) why is Super_scaffold_65 backwards? Negative gap lengths ChLG 2 super! scaffold ChLG 2 super! scaffold BNG consensus maps BNG consensus maps ChLG 2! scaffolds 133 134 132 129 135 127 136 137 139 138 140 141 142 143 144 145 Two new super scaffolds were created and the sequence similarity is being evaluated min confidence 10 scaffold_133 aligns but is not visible in IrysView?) why is Super_scaffold_65 backwards? ChLG 2! scaffolds 130 131 133 134 132 129 135 127 136 137 139 138 140 141 142 143 144 145 U 18 14 16 19 20 21 22 23 24 25 26 27 28 30 BNG consensus maps U 18 14 16 19 20 21 22 23 24 25 26 27 28 30 BNG consensus maps
  • 36. Gap lengths Distribution of gap lengths for automated output Gap length (bp) This negative alignment also indicated a potential assembly issue Count −1500000 −1000000 −500000 0 500000 1000000 0 5 10 15 20 Negative gap lengths Positive gap lengths
  • 37. Negative gap lengths This negative gap length is from a BNG consenus map joining in silico 81 and 102 and 103 Half of scaffold_81 aligns with ChLG7
  • 38. Negative gap lengths Half of scaffold_81 aligns with ChLG7 79 80 81 82 83 Because the other half of 81 aligns to another BNG consensus map that aligns to its chromosome linkage group this alignment was rejected and stitch was re-run ! The BNG maps suggest a mis-assembly of in silico 81 at a sequence level
  • 39. Distribution of gap lengths for automated output Gap length (bp) Count −1500000 −1000000 −500000 0 500000 1000000 0 5 10 15 20 Negative gap lengths Positive gap lengths Gap lengths All extremely small negative gap lengths, < -40,000 (bp) (shaded), were independently flagged as potential sequence mis-assemblies to be checked at the sequence-level
  • 40. Distribution of gap lengths for automated output Gap length (bp) Count −1500000 −1000000 −500000 0 500000 1000000 0 5 10 15 20 Negative gap lengths Positive gap lengths Gap lengths All gaps from the shaded regions were also manually rejected and stitch.pl was rerun without them for the current super-scaffolded assembly ! We suspect extremely small negative gap sizes may be useful in locating sequence mis-assemblies
  • 41. Tribolium super-scaffolds Input file N50 (Mb) Number of Contigs Cumulative Length (Mb) genome FASTA 1.16 2240 160.74 super-scaffold FASTA 4.46 2150 165.92 N50 of the super-scaffolded genome was ~4 times greater than the original ! Super-scaffolds tend to agree with the Tribolium genetic map
  • 42. Tribolium super-scaffolds Input file N50 (Mb) Number of Contigs genome FASTA 1.16 2240 160.74 4.46 2150 165.92 For Tribolium : first minimum percent aligned = 30% first minimum confidence = 13 Cumulative Length (Mb) second minimum percent aligned = 90% second minimum confidence = 8 ! super-scaffold FASTA Lower quality alignments were manually selected if genetic map also supported the order Complex scaffolds were broken manually for sequence level evaluation
  • 43. Tribolium super-scaffolds min confidence 10 From ChLGX, 11 of the previous 13 scaffolds were joined with two unplaced scaffolds (U) into one super scaffold. ChLG X was reduced from 13 scaffolds to 2 with one scaffold being moved to ChLG 3 ChLG X super! scaffold BNG consensus maps ChLG X! scaffolds BNG consensus maps U 3 4 5 6 7 U 8 9 10 11 12 13
  • 44. Tribolium super-scaffolds min confidence 10 51 U 43 45 44 46 The second scaffold from ChLG X aligned to scaffolds from a portion of ChLG 3 ChLG 3 super! scaffold BNG consensus maps ChLG 3! scaffolds BNG consensus maps 32 33 34 35 36 2 37 38 39 40 41 42 ChLG 3 super! scaffold BNG consensus maps ChLG 3 super! scaffold BNG consensus
  • 45. Tribolium super-scaffolds min confidence 10 From ChLGX, 11 of the previous 13 scaffolds were joined with two unplaced scaffolds (U) into one super scaffold. Two unplaced scaffolds aligned to ChLG X ChLG X super! scaffold BNG consensus maps ChLG X! scaffolds BNG consensus maps U 3 4 5 6 7 U 8 9 10 11 12 13
  • 46. Tribolium super-scaffolds min confidence 10 From ChLGX, 11 of the previous 13 scaffolds were joined with two unplaced scaffolds (U) into one super scaffold. 4% Redundancy in alignment may be from assembly of haplotypes (generally observed as two BNG consensus maps aligning to the same in silico map) ChLG X super! scaffold BNG consensus maps ChLG X! scaffolds BNG consensus maps U 3 4 5 6 7 U 8 9 10 11 12 13
  • 47. Tribolium super-scaffolds overlapping BNG cmap min confidence 10 From ChLGX, 11 of the previous 13 scaffolds were joined with two unplaced scaffolds (U) into one super scaffold. ChLG X super! scaffold BNG consensus maps ChLG X! scaffolds BNG consensus maps U 3 4 5 6 7 U 8 9 10 11 12 13
  • 48. Tribmoinl icuonmfid esnucep 1e0r-scaffolds ChLG9 currently (2150 scaffold_133 aligns but is not visible in IrysView?) why is Super_scaffold_65 backwards? 128 130 131 133 134 132 129 135 127 136 137 139 138 140 141 142 143 144 145 For ChLG 9 21 scaffolds were reduced to 9 ChLG 9 super! scaffold BNG consensus maps ChLG 9! scaffolds BNG consensus maps
  • 49. min confidence 10 Tribolium super-scaffolds For ChLG 5 17 scaffolds were reduced to 4 ChLG 5 super! scaffold BNG consensus maps ChLG 5! scaffolds BNG consensus maps 69 68 70 71 72 73 74 U 75 76 77 78 79 80 81 82 83
  • 50. Acknowledgements K-INBRE Bioinformatics Core! Susan Brown - PI Nic Herndon - script development Nanyan Lu - manual editing Michelle Coleman - extractions and running the Irys! Zachary Sliefert - metric summaries ! Bionano Genomics! Ernest Lam - assembly pipeline best practices assistance Weiping Wang - assistance with data formats Palak Sheth - collaboration to standardize analysis ! Script availability! https://github.com/i5K-KINBRE-script-share/Irys-scaffolding BNG scripts available by request from BNG
  • 51. Gap lengths Distribution of gap lengths for automated output Gap length (bp) Of the automated stitch.pl Tribolium super-scaffolds there were 66 gaps had known lengths and 26 had negative lengths (set to 100 (bp)) ! Of the manually edited Tribolium super-scaffolds there were 66 gaps had known lengths and 24 had negative lengths (set to 100 (bp)) Count −1500000 −1000000 −500000 0 500000 1000000 0 5 10 15 20 Negative gap lengths Positive gap lengths