1. Denovo genome assembly of Moniliophthora roreri
Group 4.
Chen, Demeke, Habte, Namrata, Rajdeep, Xu

2. Introduction
M.roreri is a fungal pathogen that causes frosty pod rot in cacao (Theobroma cacao) mainly in central and south America
Genomic information is important to enhance our understanding of the pathogen biology
Genomic assembly is more important and challenging when there is no reference sequence

3. Assembly Pipeline Gap filling (Gap filler) Gene Prediction (Quast)
Quality Control
（FastQC)
Scaffolding
(SSPACE)
Adapters Remove
(Trimmomatic)
Contaminant cleaning
(Bowtie)
Contig assembly
(Minia)

4. Introduction to minia An ultra-low memory DNA sequence assembly
Human genome can be assembled using 4 GB of memory
Produces results of similar contiguity and accuracy to other de Bruijn assemblers like velvet
Takes set of short genomic sequences (typically - Illumina DNA sequencer)
Version used: Minia maxk128

5. Recommended k-mer based contig assembly
K-mer estimation (Kmergenie)
Minia assembly
Library
Recommended K
Minimum coverage
Predicted
assembly size
N50
Longest
# Contigs
Total length
PE reads 77 4
55,981,704
5879
54,716
14,236
47,131,570
MP reads 93 7
56,496,136
1384
21,495
35,033
43,387,625
Unpaired reads 71
56,004,620
7431
54,409
11,989
45,327,642
PE and MP reads 87 16
57,157,776
6488
45,213
13,355
48,323,321
All 11
56,949,824
4017
35,607
18,455
48,488,928

6. Optimizing the k-mer selection for final assembly
All library - all (PE, MP and unpaired)
Minimum coverage set to 4
K-mer size
Minia assembly
N50 (bp)
Longest (bp)
# Contigs
Total length (bp) 51
16,767
187,673
10,050
47,937,871 61
18,316
255,017
9,593
50,039,743 71
19,720
189,801
9,008
51,488,056 81
20,068
155,025
8,476
52,624,232

7. Effect of k-mer, data type on the assembly
Data used
k-mer
Abundance threshold
N50 (kb)
Longest Contig (kb)
# Contigs
Assembly length (Mb)
All 81 9
19.7
155.0
8595
52.4 12
18.8
147.9
8571
51.4 19
17.3
114.2
8458
48.2

8. Scaffolding: SSPACE used
Standalone scaffolding program
Extend and scaffold pre-assembled contigs
Uses Bowtie to map paired libraries to a pre-assembled contigs
Use positions and orientations for scaffolding
Pairs are found within the allowed distance
Together with their orientations - used for contig pairing & ordering

9. Effect of the library and insert size on scaffolding
Library (insert size)
# scaffolds
N50 (kb)
Longest Scaffold (Mb)
Total Length (Actual sequence)
Ns/100 kb (Total Ns)
MP (3500)
899
217.2
1.32
65.1 (52.4)
19.5 kb
(12.7 Mb)
PE (400)
3417
50.6
0.58
52.24 (52.23)
10.25
(5354 b)
MP (2500)
763
233.3
1.91
58.4 (52.4)
10.3 kb
(6.02 Mb)

10. Introduction to GapFiller v 1.11 (Boetzer et al 2012 Genome Biology)
Close gaps within previously created scaffolds
Gaps within scaffolds are defined as unknown nucleotides (N's)
the unknown nucleotides are filled with true nucleotides in order to (try) close the gap

11. Gap filling pipeline 1st cycle of gap filling 3 iterations
# scaffolds = 763
Total Ns: 737 kb (1280/100 kb)
Total length (with/without Ns): / Mb
N50: Kb
Longest scaffold: 1.90 Mb
Gaps closed : = 3481
1st cycle of gap filling
3 iterations
PE and MP libraries
# scaffolds = 459
Total Ns: 892 kb (1546/100 kb)
Total length: / Mb
N50: Kb
Longest scaffold: 3.76 Mb
2nd cycle of scaffolding
with MP libraries
# scaffolds = 459
Total Ns: kb (1138/100 kb)
Total length: 57.9 / 57.2 Mb
N50: Kb
Longest scaffold: Mb
Gaps closed : = 215
2nd cycle of gap filling
8 iterations
PE and MP libraries

12. Gene Prediction using quast

13. Summary Fairly good genome assembly pipeline
Longest N50 and scaffold, 3.7MB
Lowest # scaffolds, < 500
Fairly low # Ns

14. Assembly: All data set; K=93; M=11; SSPACE
Statistics without ref.
Minia
1st Scaffold
2nd Scaffold
# contigs
18455
831
472
# contigs (>= 1000 bp)
13394
734
410
# contigs (>= bp)
290
225
Largest contig
35607
Total length
T. length (>= bp)
T. length (>= bp)
N50
4017
182599
320808
L50
3663 76 47
GC (%)
46.71
46.81
46.8
# N's

15. Thanks