Fig 1.
The MutAid pipeline can be run with a single command. Sanger sequencing data analysis has one start point and the flow of analysis runs from top to the bottom, illustrated by a black arrow. NGS has three starting points: 1) raw reads (red color), 2) high quality FASTQ file (blue color)—in this case first step is skipped and 3) mapped reads in BAM or SAM file format (green color)—in this case step 1 and 2 are skipped. * Step is optional.
Table 1.
MutAid variant cross-referencing.
MutAid constructs direct links to more than 30 publically available databases for each variant in the output summary table. These links are created based on coordinates, and the Entrez gene ID. The table lists, which links will be created for known and novel variants, in exonic and intergenic regions.
Table 2.
MutAid variant summary output table description.
MutAid produces a final variant summary with one line per variant including experimental information, patient information, variant information, variant effects and database cross-references.
Fig 2.
Venn diagrams of called SNVs in MutAid by four variant callers with BWA, Bowtie2 and GSNAP mapping (A) Freebayes (B) GATK-HaplotypeCaller. (C) SAMTOOLS and (D) VarScan2.
GATK shows 93.29% overlap between at least two mappers whereas Varscan2 shows least overlap among all four variant callers with 78%. SAMTOOLS and Freebayes show 92.23% and 88%, respectively, agreement with at least two mappers.
Fig 3.
Venn diagrams of called INDELs in MutAid by four variant callers using BWA, Bowtie2 and GSNAP mapping results. (A) Freebayes (B) GATK-HaplotypeCaller. C) SAMTOOLS and (D) VarScan2.
GATK shows 90.78% overlap between at least two mappers and SAMTOOLS shows least overlap among all four variant callers with 74.34%. Varscan2 and Freebayes show 76.56% and 83.70%, respectively, agreement with at least two mappers.
Fig 4.
Venn diagrams of called SNV by four variant callers using (A) BWA (B) Bowtie2 (C) GSNAP with same mapper.
Result shows that 75% - 84% SNVs are common with at least two out of four variant callers. With all 3 mappers Varscan2 identified novel SNVs from 16% - 24%.
Fig 5.
Venn diagrams of called INDEL by four variant callers using (A) BWA (B) Bowtie2 (C) GSNAP with same mapper.
Consistent with SNV results more than 78% INDELS are identified by at least two variant callers.
Fig 6.
Visualization of SNVs in IGV called by MutAid pipeline with Illumina and Sanger sequencing data analysis.
MutAid produces BAM files for NGS and Sanger, which can be loaded into IGV to view and confirm the identified variants. In blue color we can see that SNV (T>C) has been identified by NGS (top panel) and confirmed by Sanger sequencing (middle panel).
Fig 7.
Visualization of conservation track in UCSC genome browser for novel variants.
MutAid constructs a direct link to the UCSC genome browser for all variants including novel variants. On top, reference nucleotides are displayed and in the bottom panel (highlighted with green color) the conservation track of several species is displayed. To confirm novel variants, conservation analysis can be performed for each mutation position. A novel mutation might be ignored if a position has poor conservation among the species (pointed by red color arrow). A novel mutation may be further analyzed if the position is highly conserved (pointed by blue color arrow).
Table 3.
Identified SNVs by MutAid pipeline.
SNVs were called using four variant callers (for each mapping result) with a minimum read coverage of 20, minimum variant allele coverage of 4 and a base quality of at least 20. The percentage given in brackets is the fraction of SNVs having an entry in the Single Nucleotide Polymorphism Database (dbSNP) version 137.
Table 4.
Identified INDELs by MutAid pipeline.
INDELs were called using four variant callers (for each mapping result) using the same settings as for SNV calling. The percentage given in brackets is the fraction of INDELs having an entry in dbSNP version 137.
Table 5.
Comparison of various features of MutAid and other tools for NGS and Sanger data analysis.