Next-Gene - Next Generation Sequencing Software

Software for Analysis of “Next Generation” Sequence data

 Download de novo assembly Application note as de novo Assembly Application Note PDF (167Kb)

 Download Digital Gene Expression Application note as Digital Gene Expression Application Note PDF (131Kb)

 Download File Format Application note as File Format Application Note PDF (98Kb)

 Download SNP & Indel Application note as SNP & Indel Application Note PDF (243Kb)

 Download Transcriptome Application note as Transcriptome Application Note PDF (605Kb)

• Compatible with all Next Generation systems
• Performs Assembly, SNP/Indel detection, Transcriptome Analysis
• “Low-end” Hardware Requirements
• Easy & Intuitive User Interface
• Exclusive SoftGenetics technical support

SoftGenetics offers NextGENe, a unique software for analyzing “Next Generation” Sequencing data, generated by platforms such as the Genome Sequencer FLX System from Roche Applied Science (454 Life Sciences) and short read systems such as the Illumina® Genome Analyzer (Solexa sequencing technology). Designed with speed and simplicity of use in mind, NextGENe is capable of completing much of the analysis on a standard PC, and soon the analyses will be available on a Linux platform. By statistically polishing the large sets of data into manageable fragments, NextGENe can quickly generate accurate results. NextGENe can be used to identify Single Nucleotide Polymorphisms (SNPs) and small Insertions and Deletions (Indels) for large scale resequencing projects and transcriptome analyses.

De novo assembly of both long and short sequences will be available shortly, as well as analysis of data from the Applied Biosystem SOLiD™ System. The software is designed to handle the unique hurtles presented by each platform, such as the homopolymers for pyrosequencing and short reads generated by both the bridge amplification sequencing by synthesis method and the sequencing by ligation method, while utilizing the system’s advantages.

Figure 1: NextGENe Alignment tool highlights SNPs and small Indels. Towards the left side, the software has identified a T>G SNP. The center of the figure shows a GAAA repeat regions, and some of the reads (highlighted in red) have the end of the repeat in the middle of the read. This scenario enables detection of both deletions, as shown here, and insertions.

 

Resequencing Projects

SNPs and small Indels can be detected in targeted sequencing data from both longer sequence reads and the short reads from techniques such as the Solexa sequencing technology. Using the Condensation Tool the short reads sometimes aligning to multiple regions of the genome can be elongated to 50 or 65 bases, increasing the probability of the sequence being unique. The Condensation Tool partitions the reads into smaller groups and sorts them into two categories, condensed fragments and fragments that are more likely to be noise from sequencing errors. Because the short reads have been elongated by the Condensation Tool, detection of Indels is possible. Since a true SNP occurs at a high frequency within the reads, the low frequency variants caused by noise can be deciphered from the positions containing a polymorphism.

 

Whole Transcriptome Analysis

Analyzing an organism’s transcriptome with the Next Generation Sequencing technology presents several challenges, including the generation of sequences with homopolymeric regions and high variability in expression rates. Short reads (25 to 35 bases) are not always unique, but fragments of 50 to 65 bases are generally unique. In addition, high expression of some genes can mask genes of low expression levels. When the sequence from a low expressed transcript is similar to the sequence of a gene expressed at a high level, this sequence could be misinterpreted as noise or error. By using the Condensation Tool, the short reads are polished into longer reads, allowing for noise and error to more reliably be filtered out.

When using the Alignment tools, the highly expressed sequences are matched to the reference. The low level reads, often mistaken as containing errors, are rescanned and matched to the reference allowing for more accurate detection of genes expressed at lower rates. Uniqueness Scores are determined for each fragment. Any Reference mRNA Sequence database can be used as a reference for alignment of the reads.

The results of the analysis can be saved as a reference file, allowing for direct comparison to the results from another analysis. This is a useful feature for comparison studies such as Chromatin Immunoprecipitation (ChIP).

Figure 2: The alignment tool shows three SNPs identified by the blue background. At the top of the figure, the first row shows the chromosome position, the vertical numbers below this show the coverage, and the reference sequence is located below the coverage. Holding down Ctrl key while cursor is in reference region at top of figure shows the annotation line.

 

Condensation Assembly Tool

The Condensation Assembly Tool is used to statistically polish and lengthen the short sequence reads into fragment sizes that are more manageable. The short reads such as those from the Illumina Genome Analyzer System are often not unique within the genome being analyzed. By clustering similar reads containing a unique anchor sequence, data of adequate coverage is condensed and the short reads are lengthened. The unique anchor sequence, or index, can be a 12 base fragment that is found in several of the reads. All reads containing this exact sequence are clustered together. Often, many of the reads within a cluster contain 4 homologous nucleotides both upstream and downstream of the index sequence. The cluster of reads can be sorted by these flanking shoulder regions into groups of similarity. The consensus of these groups is much larger in length, and often these 50 to 65 base pair fragments are unique within the genome, with exceptions such as homopolymeric regions, repeats and duplications.

Figure 3: The Condensation Assembly tool clustered similar reads containing the same anchor sequence of TCACGACGGTCT. The right shoulder, four nucleotides to the right of anchor, is divided into two sequences, AATC (red) and AACC (blue). A consensus sequence is generated for these groups.

 

Figure 4: The Condensation Assembly tool generates a fasta file of the sequence reads that were condensed into larger fragments. The anchor, index, is common sequence between the group of reads and by dividing the clusters of reads by their shoulder sequences, a longer consensus sequence is generated for the group.

 

Alignment Tools

The Alignment tools are designed to match the sequence reads to a user-defined annotated reference sequence. Once the reads have been aligned to the reference, SNPs and Indels are highlighted for quick identification. The display shows the reference sequence, aligned sequence reads, breakpoints between genes, coverage, and with the click of a button, biological information for the position can be displayed. The projects and reports can be saved for further analysis.

Figure 5: Coverage is indicated by gray lines at the top of this alignment tool. The red lines indicate the break points between the transcripts of this reference sequence file. An Indel was highlighted by the tool in the middle of the screen, and two substitutions were detected to the right.

 

Recommended Hardware

Customizable Desktop PC
• Windows Vista (64-bit)
• Intel(R) Core(TM) 2 Quad processor Q6600 (2.4GHz)
• 8GB DDR2-800MHz dual channel SDRAM (2x1024)
• 512MB NVIDIA GeForce 8500GT, TV-out, DVI-I, HDMI

 

 

Next-Gene - Software for Analysis of “Next Generation” Sequence data