Human Transcriptome RNA-Seq Analysis Using MacVector

With MacVector Pro and Assembler you can use Bowtie to perform RNA-Seq analyses using NGS data. The interface even has specialized output tabs listing the coverage information and statistics for each annotated CDS and gene feature on the genome. There is an example tutorial in the /Applications/MacVector/Documentation folder called “RNASeq Expression Analysis Tutorial.pdf” that illustrate the analysis using a small (1.6 Mbp) prokaryotic genome.

What surprises many people is that the combination of MacVector and modest Macintosh hardware can actually perform this analysis on the human genome. Now there are limitations to this – it’s not currently practical to do this with the entire genome due to memory and processing constraints, but it is possible to run an analysis against the known Human Transcriptome. The latest version of this can be downloaded from the GENCODE database. There is a new RNA-Seq Human Transcriptome Analysis Tutorial that describes the basic procedure in detail and some sample data that can be downloaded. The end result is that you get a table similar to that shown below that can be copied and pasted into Microsoft Excel for additional analysis.

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Use a right-click in the Editor tab to see if your contig can be circularized

MacVector incorporates no less than THREE different de novo assemblers, phrap, velvet and SPAdes. While all are great assemblers, with each having their own specific advantages, none of them will generate a circular sequence from input reads. However, MacVector also includes a tool to help you with this. If you are assembling reads representing plasmid sequences, or if you are closing gaps in a circular genome, you can find out if a contig can be circularized by double-clicking on it in the Assembly Project and then right-clicking* in the Contig Editor to bring up a context-sensitive menu.

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The algorithm looks for a perfect overlap between the ends of at least 20 bases. If no overlap exists, the menu item is greyed out and reads “Cannot Circularize Consensus”. Otherwise it indicates the length of the overlap. If you select the menu item, a new sequence window opens containing the circularized consensus of the contig, with all gaps removed.

*To right click with a trackpad hold down [CTRL] and click once or tap with two fingers. MacVector has many “right click” menus with extra functionality.

Not sure if you have Assembler? Choose MacVector | About MacVector. If the screen that appears says “MacVector with Assembler, Pro Edition” then you have it. If not, you can sign up for a fully functional 21 day trial version.

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Import Multi-Sequence Genbank Files into an Assembly Project for easy access to Features

There are many genomes in the Genbank database that cannot be downloaded as single annotated sequences. These might be large multi-chromosome eukaryotic genomes, but, increasingly, partially sequenced bacterial chromosomes where the major contigs have been annotated using the NCBI annotation pipeline. Typically, when you encounter these, there are options to download annotated versions of these as multi-sequence Genbank formatted files. MacVector has the option to open any file containing multiple sequences as either a Multiple Sequence Alignment document or as individual Sequence documents. This is not always optimal if you have more than a handful of sequences in the file. However, if you use MacVector with Assembler, you can import these sequences into a project using the Add Ref toolbar button and the individual sequences will not only be displayed in the project window, but, if you double-click on one, the complete annotated sequence will be opened.

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This is a great way to view and/or sort collections of annotated sequences in Genbank format that cannot be done directly through the Apple Finder. Once opened, you can Export… any sequence in another format if you wish.

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Opening Genbank or FASTA files with multiple sequences as individual sequences

Many sequence formats contain multiple concatenated sequence entries. For example FASTA and Genbank are two formats capable of storing multiple individual sequences.

By default MacVector will treat such sequences as alignments and open them in the Multiple Sequence Alignment editor. Most users who want to open such a file do want to see an alignment. Additionally if the default behaviour was to open as individual sequences, then accidentally clicking on a large alignment would result in many hundreds of individual sequence windows opening up on your desktop (do remember that holding down the OPTION key and clicking on the close button will close all open sequences).

If you need to open such a sequence file as individual sequences, then there’s a simple option that you need to check in the FILE | OPEN dialog. This behaviour has not changed for quite some time. However, a few versions back the appearance of the dialog changed, due to a change in Apple’s guidelines on file dialogs. Whereas the older dialog had an obvious way to see this dropdown menu, now all you see is a small OPTIONS button in the bottom left hand corner.

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To open multiple sequence files as individual files you need to check an option in the FILE | OPEN dialog.

  • Click FILE | OPEN
  • In the dialog click OPTIONS (bottom left corner)
  • Change OPEN MULTIPLE SEQUENCE FILE AS from AUTO to SINGLE SEQUENCES
  • Click OPEN
  • (Read More…)

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    Optimizing Align To Folder Parameters for use with NGS Data

    You can use the Database | Align To Folder function to scan large fasta or fastq files containing NGS data to find and retrieve just those reads that match a specific target sequence. The search is aware of paired-end reads, so when you retrieve hits, both reads of a pair will be saved into a pair of fasta or fastq files, even if only one of them matched the query sequence. This is a great way of finding sequencing reads to extend any short sequence. For optimum performance, set up the Align To Folder search like this.

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    Set the Search Folder to the location of your data and select the Folder contains paired-end reads checkbox if you are working with paired data. For speed, make sure Hash Value is set to the maximum (currently 12) and use a large Scores to Keep value to make sure you can retrieve all the hits. Finally, use the DNA identity with penalties matrix to optimize the search so that only very close matches are reported.

    On a moderate machine (e.g. a three year old 2.7 GHz i7 MacBook Pro), a search of 20 million x 90nt reads with a 500 bp search sequence might take from 45 mins to two hours, depending on the number of hits encountered. To retrieve the hits, select the numbered rows in the Folder Description List results tab and choose the Database | Retrieve to File… menu item. If you used paired-end data, two files will be produced, –1.fastq and –2.fastq, that you can then use as input to Assembler for SPAdes or Velvet assembly, or to Analyze | Align To Reference for more detailed reference alignment analysis.

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    MacVector and macOS Catalina

    The next OS release for the Mac will arrive during September. macOS Catalina is a major OS release and includes many new features.

    As usual our developers have been hard at work ensuring that when macOS Catalina is released, MacVector 17 will be fully compatible.

    For older versions you can check compatibility on our website as soon as macOS Catalina is officially released.

    For versions of MacVector released over the past few years it is likely that they will work fine. For these we have been striving to future proof MacVector for new versions of macOS.

    However, for older versions of MacVector there will be issues. More significantly MacVector 13.5 and all older versions will not run. The MacVector application icon will be displayed with a “stop sign” indicating it will not run.

    This is due to Apple moving fully to a 64 bit operating system.

    Starting with the release of macOS High Sierra 10.13.4 any 32 bit application would periodically display warning dialogs. For example: “the application is not optimised for this release and needs to be updated” and “This application will not work on future macOS releases”. However, the application would still run. But with the release of macOS Catalina this migration is now complete and 32bit applications will no longer run.

    Here at MacVector we always strive to stay ahead of the game. When Apple recommended (many years ago) that all applications should be 64 bit, we immediately started working to move MacVector to be fully 64 bit resulting in the release of MacVector 14 in 2015.

    Please do note that whereas our developers do develop and run MacVector on Apple’s public and developer betas of macOS, we really do not recommend that users run MacVector for any real workflows on beta releases. There may be unexpected issues that you may encounter.

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    Upgrade to the most feature packed version of MacVector yet and install on multiple Macs with a 50% discount.

    Personal licenses are ideal for using on a single Mac, but not if you have multiple Macs or want to install on a shared lab computer as well as your personal Mac. So why not upgrade to a standard license of MacVector Pro 17 that you can share among a group of users in the same research group?

    What’s even more flexible is if you take your laptop home, or away on travel to a conference, then you can work on MacVector and the license back in the lab will still work!

    During August we have a 50% discount on either exchanging a current personal license to a standard license, or upgrading an older personal license to a standard one of MacVector 17.

    MacVector 17 is one of the most feature-packed updates that we have ever released. Highlights include an interactive Restriction Enzyme Picker, a unique genome comparison tool and a tool to help you design and document Gibson Assembly and LIC workflows. MacVector 17 has support for Dark Mode, helps identify genome sequencing errors, automatically displays primer binding sites, maps multiple sequencing datasets against a single reference and has numerous performance improvements.

    How to upgrade a personal license to a standard license.

    Personal Licenses are locked to a single Mac. They are best suited to a single person using MacVector.

    Standard licenses may be installed on ALL Macs in a lab with one concurrent user. Standard licenses use Bonjour to check if the serial number is in use. If there is no network they just work.

    To upgrade request a quote or email for more information.

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    The MacVector Team will be at ASM Microbe 2019 in San Francisco.

    We’re at ASM Microbe 2019 in San Francisco from Friday 21st June until Sunday 23rd.

    The show is finally back on the West Coast at the Moscone Convention Center after 6 years on the East Coast and New Orleans.

    This year the exhibit hall hours are unchanged from last year’s show:

  • Friday, June 21st – 10:30 AM – 5:00 PM
  • Sat. June 22nd – 10:30 AM – 5:00 PM
  • Sunday, June 23rd – 10:00 AM – 4:00 PM
  • We’re on booth 1731.

    Please do drop by. We’ll be demoing our latest release, MacVector 17.

    If you’ve never used MacVector before, or you are a power user, then please drop by and say hello. We always enjoy meeting users and we guarantee we can teach you something new, and hopefully you’ll be able to teach us something new too!

    See you in San Francisco!

    The Twitter hashtag looks to be #ASMMICROBE2019.

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    What can MacVector do for my lab?

    Here’s what MacVector can do for your lab.

    Comparing sequences

    Whatever type of alignment your sequence needs, there’s a tool in MacVector.

    Cloning

    CRISPR Indel Analysis: Identify insertions and deletions following CRISPR editing of a target.

    Compare Genomes: Compares two related annotated genomes to identify identical, similar and weakly similar features.

    Sequence assembly of NGS data against a reference genome or compare your sequencing against your new construct.

    Coverage Tab: Compare different datasets assembled against the same reference sequence with expression level comparison.

    Translated Multiple Sequence Alignments: Align DNA sequences based on their translations.

    Align proteins against a reference great for comparing known proteins against an unknown one.

    Auto Annotation of common plasmid features to blank sequences.

    InterProScan: Scan proteins for functional domains against many databases.

    Cloning

    How Do I?: new menu shows common workflows with step by step guides. Every tool has a link to a video tutorial.

    MacVector

    Design Cloning workflows

    As simple as dragging a fragment to a cloning vector.

    Flexible Cloning Subclone with restriction enzymes, Gibson cloning, Gateway and more.

    Cloning history Every step is documented.

    Agarose Gel: run out digested sequences. Easily identify site(s) to differentiate successful clones.

    Restriction Enzyme Picker: easy identification of useful enzyme cut sites.

    Cloning

    Gibson/Ligase-Independent Assemblies: Automatic primer design and assembly for Gibson Assembly or Ligase Independent Cloning workflows.

    Primer Design

    Design primers with ease.

    QuickTest Primer changes primer design. Hairpin? Nudge your primer until it goes.

    Add tails to your primers with silent restriction sites/mismatches and view reading frame changes.

    Quickly design pairs of primers click a region to get the best primer pairs to amplify it.

    Scan For… Missing Primers: utilize the power of the Primer Database to display binding sites from your lab’s primer collection

    Read More…

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    HOW DO I video guides to common molecular biology workflows

    MacVector has a wide array of different tools for working with protein and DNA sequences. MacVector has always been designed with the Mac’s simplicity in mind and getting started with simple tasks is quick. However, making the most of the many functions and getting familiar with MacVector’s wide range of tools does require more help

    Nobody has time to read manuals nowadays and so with that in mind we set out to try to help new users get up to speed quicker and help more advanced users know tools they may not be that familiar with.

    MacVector 17 introduces a new menu which lists common workflows that a molecular biologist may need. Each topic has a short video and/or a short step by step guides. What’s more is every tool’s dialog now has a link to a video tutorial.

    So if you need to know how to do something then try the HOW DO I menu.

    If you need to know more about a tool then click the (?) button in every dialog.HOWDOI IndexHOWDOI MENU

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