General musings from the MacVector team about sequence analysis, molecular biology, the Mac in general and of course your favorite sequence analysis app for the Mac!

Searching and downloading sequences from Entrez

MacVector has integrated connectivity to the NCBI BLAST and Entrez databases. You can directly search Entrez for DNA or Protein sequences based on features, authors, keywords etc and directly download them into MacVector, complete with all features and annotations.

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Eastern Great Lakes workshop tour in February

The MacVector team will be touring the Eastern Great Lakes for a series of workshops in February.

We will be running workshops in Rochester NY, Buffalo NY, Ypsilanti MI, Cleveland OH, Wooster OH, Columbus OH and Cincinnati OH.

Monday, Feb. 5th

  • 10:00 – 12:00 University of Rochester, Rochester, NY
  • 2:00 – 4:00 University at Buffalo, Buffalo, NY
  • Tuesday, Feb 6th

  • 10:00 – 12:00 Wayne State University in Detroit, MI
  • 2:30 – 4:30 Eastern Michigan University, Ypsilanti, MI
  • Wednesday, Feb 7th

  • 10:00 – 12:00 Cleveland Clinic Foundation, Cleveland, OH
  • 2:00 – 4:00 College of Wooster, Wooster, OH
  • Thurs. Feb. 8th

  • Nationwide Children’s Hospital in Columbus, OH.
  • Friday, Feb 9th

  • 11:00 – 1:00 Cincinnati Children’s Hospital Medical Center, Cincinnati, OH
  • In workshops we try to highlight the new functionality introduced over the last few years to MacVector that even more experienced users may not be familiar with. The format is very informal and participants are encouraged to ask questions and help direct the workshop towards areas of the most interest. We’ve found that every workshop we have run has helped users make the most of MacVector.

    (more details to follow).

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    Using MacVector’s Auto Annotate tool to annotate blank sequences.

     

    Have you ever been sent a plain unannotated sequence, or downloaded a sequence from Entrez and been disappointed as it doesn’t have the carefully curated graphical appearance of your favorite genes? Auto annotation solves both of these common problems. The basic idea is that you can scan the sequence against a folder containing a collection of existing annotated sequences and MacVector will find the matching features in the folder and add those to the starting sequence.

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    Adding a primer to MacVector’s Primer Database.

     

    MacVector’s Primer Database tool makes it easy to store your lab’s regular primers. It allows you to save and retrieve primers from the Primer Database from the Primer3 and Quicktest Primer tools. You can also scan sequences for potential primer binding sites. The Primer Database will also store tails and allow you to specify the maximum mismatches allowed. The tool comes with a starter database of primers, but you can use existing subsequence files or import primers from Excel.

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    RNASeq Expression Analysis with NGS data

    If you have the Assembler module, MacVector can align millions of NGS reads from RNASeq experiments against large genomes and generate a coverage table displaying the relative expression levels of every gene in a genome. The key to this functionality is that you must have a reference genome with genes annotated as CDS or gene features – then you can use Bowtie to rapidly assemble reads from fastq files against the genome and generate a report listing the coverage for each feature. The steps to do this are

  • Use File | New | Assembly Project to create a new project.
  • Click on the Add Ref toolbar button to add a suitable annotated reference genome.
  • Click on the Add Reads toolbar item to add your RNASeq reads.
  • Click on the Bowtie toolbar button to assemble the reads against the reference genome.
  • Double-click on the resulting reference contig item and switch to the Coverage tab.
  • You will get a display similar to this.

    NewImage

    This lists each CDS and gene feature, along with the number of reads that aligned to each feature and the Reads Per Kilobase of transcript per Million mapped reads (RPKM) and Transcripts Per Kilobase Million (TPM) values that can be used to compare expression between genes and runs.

    There is an RNASeq Expression Analysis Tutorial available that describes this functionality in more detail. If you have updated to MacVector 16.0.4 you will find this in the /Applications/MacVector/Documentation/ folder. If you do not have 16.0.4, you can download the tutorial and sample dataset. Although this is a small dataset designed for rapid analysis, you can use this approach to align 50 million+ reads against the entire human transcriptome on a modest (16 GB RAM) MacBook Pro.

    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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    Annotating primers to your sequence with MacVector

    Designing a pair of primers to amplify a single feature is pretty quick with MacVector. Once you have designed a pair of primers with MacVector, you can quickly annotate both the primers and product to your sequence template. The annotation contains a timestamp and the primer’s characteristics. It also contains the full sequence of the primer, which means any custom ends (with mismatches or new restriction sites) are also included.

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

    MacVector 16 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 16 also includes a new feature 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.

    NewImage

    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.

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    MacVector are at the ASCB – EMBO 2017 meeting in Philadelphia

    The MacVector team will be at the ASCB/EMBO 2017 meeting this coming Sunday.

    The conference starts on Sunday the 3rd of December and runs until Tuesday the 5th. It’s being held at the Pennsylvania Convention Center.

    Our booth is 1002. If you are in Philadelphia for the conference please pop along and say “hi!”. We enjoy meeting long time users and learning how they use MacVector. You might be able to teach us some new tricks! We also enjoy meeting new users and demonstrating just how MacVector can make life easier for you in the lab! Come learn about our new Scan for… Missing Features tool. We’ll show you how you can go from a completely blank sequence to a beautifully annotated plasmid map in just a few steps. We’ll be demoing other new and old features.

    Booth hours are Sunday, Monday and Tuesday from 9:30 to 4:00 pm.

    Scan for Missing Features workflow v2

    Follow the conference on Twitter with the hashtag #ASCBEMBO2017. Follow the MacVector team on Twitter for tips, tutorials and more.

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    Downloading hits from the MacVector BLAST Map results tab

    MacVector’s BLAST Map results tab (added in MacVector 15.5) is a unique interface for examining the annotations around hits to a query sequence. Each pane in the display represents a High Scoring Segment Pair, as seen in the BLAST Aligned Sequence tab. At the lower left corner of each pane is a Download button – when you click on it, a popup menu appears with several options.

    NewImage

    The major options are to either download the entire genome, remembering this might be many Mbp in size, or to just download the displayed fragment. In general, MacVector displays the High Scoring Segment Pair region of the sequence, plus 2kb on either side to provide context, and that is what will be downloaded. To reduce the number of mouse-clicks when scrolling through a lot of results, Download to Last Folder places the downloaded sequence in the last folder that you selected (or your Downloads folder if you’ve never selected one before).

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    Turning on/off the SCAN FOR missing Features and ORFs

    If you’re running MacVector 15.5 or later then you will have noticed extra features annotated to your sequences. These are from the Scan for ORFs tool (added in MacVector 15.5) and the Scan For Missing Features (added in MacVector 16) tools that automatically scan every DNA sequence window for open reading frames, missing annotations and displays the results in the Map tab.

    It’s very useful, especially if you frequently receive blank sequences from colleagues, or you want to show quickly show coding frames in unannotated trace files you receive from your sequencing service. However, if you are working with richly annotated sequences, you may find these distracting.

    It’s easy to turn these off (and back on again). Both tools are controlled in the MacVector | Preferences -> DNA Map pane, along with the Scan For Restriction Sites, settings. MacVector will remember this setting for all new sequences you open, until you turn it back on again.

    The appearance of the ORFs can be controlled using the Options | Default Symbols menu item.

    Future releases of MacVector will see more automatic annotation tools, so if there’s something you’d like to see please email MacVector Support.

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