Apple started transitioning their processor architecture from PowerPC (PPC) to Intel’s x86 in 2006. The Macbook Pro and the iMac being the first Intel Macs. When they did so they gave Xcode (the OSX development environment) the ability to create Universal Binary application. These contain native executables for both PPC and x86 CPUs. So giving these applications the ability to run natively on both types of Mac.

However, to support earlier applications that had only been available as PPC executables they included a PPC Emulator called Rosetta (named after the Rosetta Stone). This allowed PPC only applications to run on Intel x86 Macs.

OS X 10.5 Leopard was the last release of OS X that was distributed as a Universal Binary OS. With OS X 10.6 Snow Leopard being a x86 only release. That is it will not run on PPC Macs. Even so they kept Rosetta as an optional install (previously it was installed by default). However, OS X 10.7 Lion does not include Rosetta and you cannot install earlier versions. This means that PPC applications will no longer run on Lion. In reality this is only applications that are over six years old.

MacVector 9.5 (released in 2006) was the first Universal Binary version of MacVector. Previous versions are PPC only and will not run on Lion.

If you are running such an old version of MacVector then your options are limited to either upgrading MacVector or not upgrading to Lion. You can still upgrade such an old version of MacVector at a significant discount. Contact Sales to request a quote or visit the website. There’s many other reasons to upgrade as well. The interface has changed substantially since MacVector 9.5 being more powerful and easier to use. There’s also many new tools with enhancements to older ones. The latest release is MacVector 12.5 and this release has improvements to sequence assembly (support for NGS reference assemblies using Bowtie) and extra options for multiple sequence alignment with Muscle and T-Coffee added.

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Both T-Coffee and Muscle are progressive alignment algorithms as is ClustalW. Progressive alignments generally build a guide tree that represents the pairwise relationships between each possible pair of sequences in the alignment. A multiple sequence alignment is then built sequentially using the tree as a construction guide. T-Coffee builds a library of all pairwise alignments but also aligns each sequence in the pair with a third sequence in the sequence set before building the MSA. Muscle does not do a pairwise alignment but instead uses an approximate method of comparing the number of short subsequences (k-mers, k-tuples or words) that each pair of sequences share. You can immediately see how this is much faster for alignments containing many sequences where the number of pairwise alignments needed to construct the tree is high.

T-Coffee is regarded as being slightly slower than ClustalW but will produce more accurate alignments for distantly related amino acid sequences. Here’s the original publication. Incidentally T-Coffee stands for Tree based Consistency Objective Function For AlignmEnt Evaluation.

Muscle is generally regarded as faster than Clustalw and T-Coffee at the penalty of being slightly less accurate.

All three algorithms are integrated into the MSA editor. This means you can try all three algorithms on the same alignment to see the results.

Here’s a few benchmark for protein sequence alignments (the files are in the Sample Files folder in the MacVector application folder:

- Clustalw: 1min26secs

- Muscle: 12 seconds

- T-Coffee: 17mins22secs

Because of the initial step of constructing the pairwise alignments tree, progressive alignment algorithms have difficulties with alignments where do not overlap at all. For example take an alignment of three sequences where you have 5kb sequence and two regions of this sequence of around 1KB long. One subsequence aligns from 1,000 to 2,000 of the 10kb sequence and the other aligns at 4,000 to 5,000. Most progressive alignments will try to create initial pairwise alignments of all combinations of sequences and that skews the alignment so that it prefers to align the sequences so that there is at least one segment of overlap between all of the input sequence. Due to not creating pairwise alignments the “Muscle” algorithm is unique amongst these three algorithms as it will align this type of data as long as the “Diagonals” optimization parameter is set to “On”.

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A consequence of this is that on Lion older versions of MacVector do not have the correct permissions to create the required MacVector License folder (Snow Leopard and earlier are fine). This seems to be OK on new Lion Macs, but it is a problem for Macs that have been upgraded from Snow Leopard or earlier, or where the user migrated their old Mac to the new one. Note that only MacVector 9.5 and later will run on Lion. Older versions will not run.

The following steps should fix it:

“[StartupHD]/Library/Applications Support/ MacVector

[StartupHD] refers to whatever name you call your hard drive.)

To modify the permissions of a folder:

Now try reactivating the license.

If this does not work then please contact Support@macvector.com.

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So after you’ve chatted about the holidays with your colleagues have a slow start to 2012 and take the opportunity to keep your copy of MacVector up to date. MacVector 12.5 was released at the end of last year. So if you’ve not yet done so then do so here! It’s also a good idea to run a few system maintenance tasks to make sure your Mac is running great.

Happy New Year!

We have a lot to look forward to in 2012. MacVector 12.6 will be released in the first quarter of 2012 with some great new primer design tools. We’ve also got another release planned for the backend of the year.

But for now, we thank you and we wish you a prosperous, happy and enjoyable New Year from the whole MacVector team.

But before we all break for the end of the year remember that we’ve just released MacVector 12.5. You’ve probably seen the release via Software Update inside MacVector but if not you can download it from our website if your license is valid for this release or download a 21 day trial.

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This is a major release with a lot of new features. Many parts of MacVector have been substantially rewritten both to keep up with OS X as well as many dialogs that have been redesigned.

The main new features are for sequence alignments. We’ve added Bowtie to Assembler for further support of NGS data assemblies. Along with variant detection using Samtools to match the existing SNP report tab.

We’ve also added additional alignment algorithms. Both Muscle and T-coffee have been added to the Multiple Sequence Alignment analysis interface, complementing the existing ClustalW algorithm. We’ve wanted both of these for a while now and judging from the results of last year’s survey so have many users.

There’s also enhanced support for segmented features as well as being able to display translated CDS features directly in the sequence editor as well as the Map view. There’s SNP reporting for the Align to Reference editor as well as greatly improved performance for its algorithm and also for the Auto Annotation tool.

MacVector 12.5 is fully Mac OS X 10.7 (Lion) compatible as well as being supported on OS X 10.5 (Leopard) and OS X 10.6 (Snow Leopard). However, MacVector 12.5 is not supported on OS X 10.4 (Tiger). If you are still using Tiger (from our last survey much less than 1% of our users still use Tiger) MacVector 12.0.6 will still remain supported.

This is just a short overview, if you have a valid license download the updater or if you do not download a 21 day trial.

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With each release of MacVector as well as adding new features we continually redesign and rewrite many existing parts of MacVector. This is done for a number of reasons. For example:

As a recent example you can now run a primer analysis and literally drag and drop a primer pair or their product to the sequence window to annotate those features to the sequence.

Here’s a few of those general improvements that have been included in MacVector 12.5.

Interface improvements

Sequence Window Enhancements

Miscellaneous Enhancements

..and remember if you want to add Assembler before MacVector 12.5 is released you can do so at half price. You can also purchase an upgrade or a new license before the release of MacVector 12.5 and get a 10% discount. Both these offers include a free upgrade to MacVector 12.5 when it is released. Please request a quote now. Don’t forget to quote the promotional code of “Assembler50%” or “MV12510″ for the discounts.

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With Assembler 12.5 our developers have come up with an affordable and straightforward solution for assembling and visualizing your NGS data. Generating sequencing data is cheaper than it has ever been, however, with this increase in data has come a problem with analysis. Assembler will now create reference assemblies with just a few mouse clicks using Bowtie. Instead of sending your millions of reads away to be assembled or delving into complicated software tools you’ll be able to align millions of NGS reads to multi megabase reference sequences in literally minutes. Bowtie is a fast algorithm, and although it’s an ungapped assembler, what it loses in accuracy it makes up for in speed. You do not need a 32GB 8core Mac Pro to assemble your data. In addition to the existing phrap/phred tools this makes Assembler a simple, cost-effective solution to analyzing your Next Generation Sequencing reads.

Creating a Reference Assembly

Your reference sequence can be in any “openable” format. However, your reads need to be in FASTQ format.

Hit Reporting

In the dialogue you’ll see an important setting called Hit Reporting. Bowtie uses a concept of strata to score alignments. A stratum is defined by all reads that contain the same number of mismatches in the seed (the seed is the first “n” bases of a read which is given higher priority in scoring than the entire read). You can either show ALL ALIGNMENTS, REPORT BEST ALIGNMENT ONLY (show the best alignment in the stratum with the least amount of mismatches) or REPORT ALL BEST ALIGNMENTS (which shows the best alignment in all strata). Which you choose depends on a few factors. For example how many references you have, how many repeated regions you expect, whether you are using a reference sequence from the same organism or a related one, and many others. Generally start with show all alignments, which is the quickest, and work from there.

Analysis

..and that’s how easy it is. Of course generating results is always easier than analysing them and to help analyse your reference contig Assembler has a few useful tools. We’ll talk about variant detection in a later blog post, but the coverage map is one of the first tools that you will see upon completing an assembly.

Using the Coverage Map

It is extremely useful to be know the depth of reads that are aligned on your reference. Areas of low coverage indicate that you need further sequencing and peaks of high coverage can be indicative of repeats. The Map view of a reference contig will show details of the depth of reads in a coverage map with four statistics. A single plot line shows a running average of the number of reads at that point. However, an average plot is not very sensitive when viewed at a high level and so two shaded areas indicate the maximum value and the minimum value of the averaged reads at that point. As the coverage map is viewed at a lower level these three values will become increasingly closer to the extent that when viewed at, or close to, residue level these three plots will become identical. Areas of zero coverage are shown in light grey. Note that these areas are always displayed even when they are disproportionate to the level of magnification.

Multiple reference sequences

You can add multiple reference sequences and depending on the settings reads will be aligned against the best match or against multiple ones. This is great for such tasks as identifying a sequenced isolate amongst a series of closely related strains of virus or bacteria. Having multiple reference sequences helps determine which is most closely related (or identical) to the isolate.

Paired end reads

Paired end reads are very useful for improving the accuracy of alignments and also for indel detection. Paired end reads are created by sequencing both ends of the same DNA molecule, with known fragment size. Since the two reads are now separated by a known distance assembly and orientation of the two reads is less complicated. For Assembler if your reads are paired end all you need to do is ensure that the same filenames but appended with version numbers and Paired End assembly is enabled.

e.g.

READS_1.fastq
READS_2.fastq

You’ll also need to input the fragment size.

In the next Assembler post we’ll talk about variant detection.

..and remember if you purchase an upgrade or a new license before the release of MacVector 12.5 you can get Assembler with a 50% discount and a free upgrade to MacVector 12.5 when it is released. This offer ends on 1st December. Please request a quote now. Don’t forget to quote the promotional code of “Assembler50%”

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However, the most important new feature in MacVector 12.5 is the addition of Bowtie to MacVector’s sequence assembly module, Assembler to create reference assemblies with your NGS data. Additionally, the Contig Editor has been enhanced with some useful features to visualise your genome sized alignments. With these improvements, our developers have come up with an affordable and straightforward solution to assembling your NGS data. Generating sequencing data is cheaper than it has ever been, however, analyzing it is not. Instead of sending your millions of reads away to be assembled at great expense, or spending time on the computer trying to assemble your reads, Assembler will now create reference assemblies with just a few mouse clicks.

To mark the release of MacVector 12.5 and this great new functionality in Assembler we’re offering users an opportunity to add Assembler to your MacVector license for half price. Not only that but if you have an older license of MacVector and want to upgrade your license then you’ll still be able to add Assembler at half price. If you do not have MacVector and want this great new functionality then you’ll still be able to add Assembler to a new license for half price.

If you take advantage of this offer, you will not only get a great price on Assembler, but you will get MacVector 12.5 and a free upgrade to MacVector 12.6 a few months after. MacVector 12.6 will include even more NGS support as well as a Quick-test function to easily analyze your existing primer sequences, and more. Plus, your license will include a year’s maintenance which will not start until MacVector 12.5 is officially released on the 1st December, 2011.

Remember that this half-price offer ends on 1st December, and the price of Assembler is going up next year. Please request a quote now. Don’t forget to quote the promotional code of “Assembler50%”

If you want to see the new functionality then we have a prerelease preview available for download. This is not yet a release candidate but is part of our external beta program

..and remember both MacVector 12.0.6 and MacVector 12.5 are fully supported on OS X Lion.

MacVector 12.5 will be released on the 1st of December, 2011

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Assembler has always made it easy to assemble your sequencing projects. It hides the complicated algorithms and provides a point and click interface to show you the results. With the release of MacVector 12.5 the range of tasks that Assembler will perform has been greatly extended. Now with the addition of high throughput reference assembly using the popular Bowtie algorithm, Assembler can now support the alignment of many millions of NGS reads against genome sized references. Assembler will also generate output in the popular BAM format. Collections of contigs can be exported in FastQ format for additional analysis. Additionally the interface has been enhanced to increase the number of reads that can be submitted for de novo assembly. ..and to analyse your assemblies SNP detection and reporting has been enhanced with VCF output from Bowtie alignments and listing of all the codon and amino acid changes between the consensus and reference sequence alignments.

Here’s a selection of tasks that Assembler will make easy for you

De novo assembly of Sanger trace files

Add in your Sanger sequencing trace files, basecall the reads to improve accuracy then assemble using quality scores.

de novo short read Assembly

Add short reads from a variety of sources in FASTQ format as well as Sanger sequencing. Great for Hybrid assemblies

Reference assembly to identify SNPs in a bacterial/viral isolate

Add a Reference sequence and an NGS file(s) representing the sequence of an individual isolate and assemble using Bowtie. View a report listing all the potential SNPs based on the differences between the consensus and the reference. Be able to quickly identify the genes that the SNPs lie in and drill down to view the nucleotide and amino acid changes.

De novo bacterial assembly assisted by a Reference scaffold

Create a reference assembly using Bowtie, then take the individual contig consensus sequences along with all the input NGS Reads that did not assemble and assemble with a de novo assembler (Phrap) directly from within a new Assembly Project.

Assembly to multiple similar references

Take reference sequences from a series of closely related strains of virus or bacteria. The reads come from a single isolate. Use Bowtie to assemble these against the collection of References to determine which is most closely related (or identical) to the isolate.

Assembly to multiple dissimilar references to identify SNPs

Essentially similar to the bacterial SNP assembly, but using yeast or some other organism with multiple genomes (or even some bacteria that have multiple chromosomes or large plasmids).

Exome (transcriptome) sequencing)

Use the genomic sequence of an organism as a reference and align reads from sequenced mRNA, cDNA or total RNA of that same organism. Uses are splice site junction identification, novel gene identification amongst many others.

..and remember if you purchase an upgrade or a new license before the release of MacVector 12.5 you will get a 10% discount and a free upgrade to MacVector 12.5 when it is released. Contact Sales@macvector.com and quote “MV12510″ for the discount.

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