Evaluation of contig sequence accuracy and the
instruction for users
We do this evaluation using the simulation data. We choose two small genomes
Arabidopsis (119 Mb) and Fruitfly (120 Mb) as the testing target, which will use less
computer time, and also be representative of both plants and animals. We simulate
100-bp paired-end reads on the reference genome, with the standard coverage (170bp,
25X; 500bp, 20X; 800bp, 15X; 2Kb, 10X; 5Kb, 5X; 10Kb, 3X; Total, 78X). The
average simulation sequencing-error-rate is 1%, the insert size is in normal
distribution and the SD (standard deviation) is 1/20 of the mean value, all parameters
are similar to the real illumina data. The advantage here is that we used homozygous
reference genome, so the heterozygotes will not confuse the count of assembly errors.
We assembled the simulation PE reads using SOAPdenovo with standard steps and
parameters: (1) sequencing error correction; (2) Contig construction (K=31 bp, do not
set the “–R” and “–M” option); (3) Scaffold building iteratively with each insert-size
data; (4) Gap-closure by local assembly of reads with one end mapped on the
neighboring contig and the other end fall in the gap. After the assembly, we aligned
the contig sequence (i.e. continuous bases, no "N") before and after gap-closure to the
reference genome sequence using LAST (http://last.cbrc.jp) with default parameters,
find the best hit locus (with highest score) for each contig, and then count the
mismatched and insertion/deletion (Indel) differences.
The result shows that the accuracy on original contigs is quite high (~99.999%),
however, the accuracy gets 1 orders down on the final contigs (~99.99%), with the
major mis-macthed and indel errors brought-in by the gap-closure steps, detailed
assembly error rate statistics is showing below. The single-base level assembly error
rate differs significantly between original contigs (1E-5) and the gap-closed regions
(1E-3). So we will label the bases in original contigs with capital characters (A,C,G,T),
and label the bases in gap-closed regions with lowercases (a,c,g,t), and we also advise
the users to be cautious on the gap-closed regions when doing gene annotation and
functional analysis.
Table. Evaluation of contig sequence accuracy using arabidopsis and fruitfly
simulation results.
Contig
Contig
Average
Contig
Contig total
Species
genome
Steps
Indel
N90
Mis-matched
N50 size
length (bp)
coverage
size
bases rate
(bp)
(%)
Arabdidopsis
length
bases
of indel
rate
(bp)
(bp)
Original contig
107,243,954
90.12%
6,559
442
2.42E-05
3.40E-07
1.17
Final contig
110,022,157
92.46%
39,282
5,208
1.85E-04
2.36E-05
3.04
(genome
size: 119 Mb)
Fruitfly
Original contig
112,687,940
93.68%
27,223
4,953
6.06E-06
6.21E-08
1.40
Final contig
113,889,393
94.68%
116,502
24,927
5.62E-05
1.07E-05
4.39
(genome
size: 120 Mb)
Notes: (1) Original contig, contigs before gap-closure; (2) Final contig, contig after gap-closure. Contig mean
continuous bases (i.e. non “N” character) inside scaffolds.