GAB-2015v6n2 - page 6

Genomics and Applied Biology 2015, Vol. 6, No. 2, 1-7
http://gab.biopublisher.ca
3
adaptors and other contaminated materials then quality
of sequence also checked with this tool and finally
high quality filter sequence file considered for de novo
sequence assembly (Table 1).
Table 1. NGS QC Toolkit Result
File Details Original File
High Quality
(HQ) Filter file
Total number of reads 12427455 12131939
Total number of bases 608945295 594465011
Percentage of HQ
reads
-- 97.62%
2. De novo Sequence Assembly
CLC GENOMICS WORKBENCH 7 considered for
de novo sequence assembly with by default
parameters like Mismatch Cost = 2, Insertion Cost = 3,
Deletion Cost = 3, Length Fraction = 0.5, Similarity
Fraction = 0.8, Word size = 21 and finally 22748
contigs generated with average value of 503 by this
software and other details are shown in Table 2.
Table 2. Contig measurement
Description Length
N75 348
N50 588
N25 1056
Minimum 197
Maximum 6080
Average 503
Count (Contigs) 22748
3. Functional annotation with BLASTX and
blast2GO
3.1 BLASTX
BLASTX was performed to align the contigs against
non-redundant sequences database using an E value
threshold of 10-6. Out of 22748 transcript contigs,
13482 were having BLAST hits to known proteins
with high significant similarity and 1114 had no
BLAST hits (Table 3). Out of total transcripts contigs,
Table 4 and Figure 1 shows that species distribution in
which 9819 sequences showed significant similarity
with Medicago truncatula and least similarity was
found with Prunus mume (24).
Table 3. Blast Result
Without Blast Results 0
Without Blast Hits 1114
With Blast Results 13482
With Mapping Results 500
Annotated Sequences 7652
Total Sequences 22748
Table 4. Blast Result of Species Distribution
Species Blast
Hit
Medicago truncatula 9819
Cicer arietinum 7942
Glycine max 1050
Pisum sativum 553
Phaseolus vulgaris 513
Lotus japonicus 168
Vicia faba 131
Vitis vinifera 118
Medicago sativa 81
Citrus sinensis 80
Cucumis sativus 74
Populus trichocarpa 73
Theobroma cacao 66
Trifolium pratense 65
Morus notabilis 60
Eucalyptus grandis 52
Prunus persica 46
Arabidopsis thaliana 46
Ricinus communis 41
Erythranthe guttata 38
Fragaria vesca 38
Jatropha curcas 38