Sequencher -Model 5.4.6 -Next-Generation DNA Sequencing (NGS) Software

Gene Codes has long been an innovator, investing in the R&D to develop powerful features for your DNA sequence analysis.  Gene Codes developed the Assemble to Reference Sequence strategy that is widely used to speed up assembly and assign base-numbering systems and features to new data.  The variance table was developed in the mid 1990`s and became a key element first for forensic sequencing of mtDNA, and then for virtually all of our collaborators. 

Working with core labs that use structured naming conventions to track data for individual clients, we developed the Assemble by Name strategy that has become such a powerful and popular tool for combining multiple sequencing projects into a single analysis run.

We have continued our strategy of adding functionality to Sequencher that focuses on labs doing DNA sequencing.  Unlike many companies in this field, we are continuing work on improving the productivity and utility of Sanger sequencing based on feedback from our collaborators around the world, and focusing on smoothly integrating those features with the latest tools for next-generation DNA sequence alignment. If you work in a lab that supports NGS, you must already have information technology [I. T.] support, but our mission is to make the data and analysis of ALL sequencing data accessible and understandable to life scientists without requiring that they have a degree in computer engineering. 

In the NGS space, trust the accuracy of your work to peer-reviewed and published algorithms and tools such as GSNAP for reference-guided alignment, Velvet for de novo assembly, Cufflinks for RNA-Seq Differential Expression and Tablet for visualization. The design and effort that goes into Sequencher makes them easy to use because once you have learned how to use one, you have learned how to use them all.

Your results are important to you. Whether you work with Sanger Sequencing or Next-Gen Sequencing, you will want to compare your results with publically available data. Sequencher Connections is ideal for a quick search or a more detailed and prolonged analysis across multiple databases and with different parameters.

Look through some of the topics at the left and explore some of the features, both common and specialized, that are in Sequencher today.

Sequencher gives you the DNA sequence editing tools you need to know that a sequence is absolutely correct. You can look at your chromatogram data one sequence at a time or view multiple aligned chromatograms in both forward and reverse orientations.

It is quick and easy to scroll through your aligned data or you can use Sequencher`s selection tools to highlight regions of discrepancy or low quality.

Automated DNA sequencers occasionally produce poor quality reads, particularly near the sequencing primer site, and toward the end of longer sequence runs. The sequences of clones from DNA libraries frequently contain vector sequence, polyA tails, or other unrelated sequence. Introns and primer sequence frequently flank the sequence of amplified exons. Unless removed by trimming, any of these artifacts will distort your sequence assembly and downstream sequence analysis.

Sequencher provides simple-to-use but powerful tools that help you trim poor quality or ambiguous data:

Trim Ends removes misleading data from the ends of sequencing fragments.

Trim Vector removes sequence-specific data contaminating the ends of your sequences.

Trim to Reference eliminates the ends of sequences that extend beyond an assembled Reference sequence.

Prior to executing a trim, Sequencher displays a graphic representation of the proposed trim, which allows you to even further refine your criteria.

Sequencher`s intuitive controls allow you to set your sequence assembly parameters and adjust them within seconds, allowing you to assemble your DNA fragments quickly and accurately. Sequencher will automatically compare the forward and the reverse-complement orientations to assemble the best possible contigs, so you can assemble DNA sequences regardless of orientation.

Apply Sequencher`s versatile assembly tools to

• Compare gene variants to a reference sequence
• Confirm vector constructs
• Assemble viral and bacterial genomes
• Cluster tens of thousands of sequences from cDNA libraries
• Assemble cDNA to genomic sequence
• Create a primer map

The Reference sequence is a powerful feature, core to many aspects of sequencing and sequence analysis. Whether you are SNP hunting, engaged in Forensics, phylogenetic studies, medical genetics or population studies you will want to use the Reference Sequence feature.

Import a sequence in GenBank format and its feature table will be applied to the sequence, and mark it as a Reference Sequence. Assembling to the Reference Sequence means you are able to compare your reads to the archetypal Reference Sequence. If you are working with multiple samples from different sources you can even use Assemble by Name to automate your work.

You can even use the Reference Sequence to guide the removal of sequences outside your region of interest or fill gaps in the sequence coverage.

Visualize the results using the Variance Table and its powerful reports.

Clustal

Clustal^[1] has been part of the Sequencher family of plugins since version 4.9. It is a widely used multiple-sequence alignment program which works by determining all pairwise alignments on a set of sequences, then constructs a dendrogram grouping the sequences by approximate similarity and then finally performs the alignment using the dendogram as a guide. You can use Clustal to align your sequences directly from the Sequencher project window. Choose from a range of parameters to control the alignment process.

Once the alignment is complete you will see the results as a contig or contigs within Sequencher which you can subject to a variety of analyses.

Speed up your Clustal alignments by combining Clustal with the power of Sequencher`s Assemble by Name functionality for aligning multiple sequences from different sources.

Finally, export the results in a variety of different formats such as MSF, Phylip, NEXUS and FastA for use in other programs or simply create a consensus and export it.

MUSCLE

MUSCLE^[2], a multiple-sequence alignment (MSA) program, joins the Sequencher 5.1 family of plugins. It joins Clustal, making it the second MSA program in Sequencher’s DNA-Seq Tools. Boasting both speed and accuracy, it compares very favorably ^[3] to other multiple-sequence alignment programs.

MUSCLE is said to have four major steps in its alignment process. The first step constructs a distance matrix between pairs of sequences using k-mer clustering, this is then converted into a tree. The second step uses this tree to guide a progressive alignment. In the final two steps, the MUSCLE algorithm tries a number of different methods to see if it is possible to improve the tree and hence the multiple alignment.

Once the process is complete and MUSCLE has built the alignment, you will see the results as a contig within Sequencher. Use Sequencher’s tools to annotate your alignment or export your alignment and place it into a special phylogenetics program.

Sequencher provides a rich set of tools for generating linear restriction maps of your DNA sequence. Filter your enzyme selection by frequency, nature of the overhang, or length of recognition sequence.

You can also specify particular vector and polylinker sequences to help you set up your cloning strategy.

Support for Confidence Values

Sequencher displays confidence and summary confidence information (if available in your DNA sequence files) in the Project window, the Sequence Editor, and the Sequence Get Info window, so you can easily monitor the quality of your data.

You can even specify cutoff ranges for your confidence values, and see those ranges by color codes.

Sequencher has several powerful tools to help you detect mutations and SNPs in your DNA sequences. You can use Sequencher for comparative sequence alignments among a group of sequences, or to compare 1 or more sequences to a Reference Sequence. Sequencher’s Call Secondary Peaks... function analyzes all of your sequences for potential heterozygotes. It’s easy to control the stringency that defines a heterozygote.

Map the positions of all heterozygotes in the DNA assembly Overview.

You can navigate from one heterozygote to the next; just click on the spacebar in the Bases view. View protein translations for both the consensus and the Reference sequences below the consensus. The Reference sequence ensures that the numbering of your SNPs is consistent from one DNA assembly to the next.

For more detailed information on finding SNPs using Sequencher see the tutorials on SNP Hunting and More on SNP Hunting.

Sequencher batch processes your DNA sequence data in a way that is transparent, user definable, and recoverable, and Sequencher never jeopardizes the validity of your scientific conclusions for the sake of automation. Sequencher always gives you the final choice in your sequence editing.

Sequencher always maintains two copies of your data, the edited and the originally imported data. You can undo all or a portion of your edits when you apply the Revert to Experimental Data command to a selection of sequences in your project or to a selection of bases in a sequence.

The Assemble by Name tool allows you to choose a portion of the fragment name to act as a shared identifier, or "assembly handle." Sequencher then makes the selections and names the contigs automatically. Sequencher even supports Regular Expression matching for setting up the unique IDs!

For example, with the click of a button you can convert 90 files, 45 pairs of forward and reverse sequences, into 45 contigs named according to your Patient IDs. A change in your sequence assembly parameters regroups your fragments, so you can assemble the contigs according to Clone ID, Date, Primer, or any other characteristic you record in your sequence names.