Next Gen Sequencing

igv_snapshot

The advances made possible by next generation sequencing cannot be overstated.  It is an exciting area of molecular biology and we are very fortunate to have 2 next generation sequencers, the MiSeq by Illumina and the MinION by Oxford Nanopore, here at Smith College.  That said, advances are constantly being made in library enrichment and preparation, and especially in the area of statistics and bioinformatics.  Thus, the links and articles below are to serve as a good start on getting information you may find helpful as you venture into this exciting area of experimentation.

Power Points

MiSeq RNA Seq and Analysis ppt by Louie Bierwert

CummeRbundTutorial

 

Nucleic Acid and Library Prep Protocols

CWZ Mini Boiling RNA Preparation

CWZ Mini Boiling RNA Preparation BCH 390 version

NEB just recently released a magnetic bead kit for mRNA isolation from whole RNA.

For most bacterial RNA applications, you will want to eliminate rRNA.  We recommend RiboZero from Illumina for this step.

MagAttract HMW DNA kit by Qiagen Cat#67563 is the recommended DNA extraction for a MinION run.  Other variations are fine.  Just want longest DNA possible.  Regardless of how you extract your DNA, you want to finish with NEB Monarch PCR and DNA cleanup kit for best library build – cat#T1030S.  G-tube fragmentation is another optimization that may create a smaller size range and better data and reads,

For Sequencing ends – NEB may come out with one but for right now it is Illumina’s Nextera Mate Pair Preparation Kit

For single cell sequencing- consider Expedeon’s True Prime;    https://www.expedeon.com/products/genomics/dna-rna-products/trueprime/trueprime-whole-genome-amplification/

Recommended Kits for Library Prep

For the MiSeq:

New England Biolabs (NEB) carries Illumina compatible DNA and RNA library prep kits and Index kits with pricing comparable to Illumina.  In addition, they will give you a substantial educational discount if using for a class.  For metagenomics, the library prep is not really kit based but uses primer design to amplify out desired discerning regions of bacteria in a sample.  See possible protocols under “16S Metagenomics.”

If you plan to run more than one sample on the MiSeq at one time (a decision made based on the genome/transcriptome size and the coverage you desire), you will need to purchase an index kit the is compatible with your library prep kit.  Only certain indexes can be mixed in order for the MiSeq to properly run and resolve your samples.  A guide to what indexes one can use together can be found here: Guide to Index combinations

NEBNExt Ultra RNA manual

Guide to Index combinations

Nextera DNA Library Prep manual

For MinION:

For DNA Oxford Nanopore’s Ligation Sequencing will give you the longest and best quality reads.  However, the Rapid DNA Sequencing kit is recommended for field or remote work.

For RNA there is a Direct RNA Sequencing kit and a Direct cDNA sequencing kit.  

There are a lot of 3rd party consummables needed for these kits so read the online manuals carefully or see Louie for help.  We have run through the Ligation Sequencing so far and have put together an in depth protocol for that one.  Will write others as experience is gained.

MinION Genomic Ligation Sequencing Protocol

 

Once your library is prepped, you will need to test it’s quantity and quality using the Qubit and the Agilent Bioanalyzer, or AATI Fragment Analyzer, each one requiring a kit based on your final product.  From those results you will then decide which reagent kit you want to purchase from Illumina for the MiSeq run based on your average library size.

Bioanalyzer DNA High Sensitivity Manual

dnf-474-hs-ngs-fragment-analysis-kit-1bp-6000bp-user-manual

dnf-474-quick-start-guide-12-capillary-11-03-2015

A Guide to Looking at Bioanalyzer and Fragment Analysis Runs

Then for the MiSeq, you need to do some calculations based on cluster density likely and amount of data needed.

Technote_Coverage_Calculation

Seq System_Coverage Calculator 2013 Aug19 (1)

Calculations And Conversions For Illumina Miseq Run

Jan2017SamplePreparationandperformingaRunonMiSeq.docx

No decisions needed for the MinION, just need a flow cell and run reagents.

The CMB is here to help with any questions you may have about making these selections. Just contact the CMB’s ITI, Lou Ann Bierwert, at lbierwer@smith.edu

MiSeq2

Helpful Literature and CMB generated Bioinformatics Workflows

All NGS

NGS-application-guide

MiSeq-Reporter-User-Guide

How to view the quality graphs and summary in BaseSpace – SequencingAnalysisViewer_UserGuide

NGS Overview and Practical Approach to Variant Detection in NGS Data – Video.  Need to download WebEx Viewer to View

MAUVE – Multiple Genome (or transcriptome) Alignment Tool – free download

 

DNA Seq

For MiSeq

Workflow DNA de novo assembly using Soap De Novo on Commandline

DNA de novo assembly using NGen on BaseSpace

Eukaryotic Genomics Assembly and Annotation on CommandLine Jellyfish, Quake, and MakerII

Eukaryote Annotation Review Article

Prokaryotic Genomics Assembly and Annotation using NextGene and RAST

For MinION-The above assemblers chop up your reads into smaller pieces negating the advantage of the MinION’s long reads.  Thus, these tools are specific for longer reads.

Canu– may want to run reads through Nanopolish first for error correction.

 

RNA Seq

Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks

RNASeq Analysis Workflow using Commandline – Tutorial

RNA Seq MiSeq Data Analysis WorkFlow Using Galaxy Mason Account_by_Louie_and_Weam

RNA Seq Analysis workflow w:o reference – Article

Advanced RNA Seq Analysis Topics and Trouble-Shooting – Article

P and q values in RNA Seq

RNASeq Analysis using Rockhopper (for bacterial transcriptomes only)

rRNAtrimNotes

RstudioTutorial

 

16S Metagenomics

16s-metagenomic-library-prep-guide-15044223-b

Workflow from sample to analyzed data – uses Qiime for analysis – Tutorial

High-Speed, Multiplexed 16S Microbial Sequencing on the MiSeq® System – Article

Qiime allows analysis of high-throughput community sequencing data – Article

Earth Microbiome Project

Examining Global Distribution of dominant archaeal populations in soil – Article

Metagenomics Research Review – Article

Ultra-high-throughput microbial analysis on Illumina platforms

Qiime Tutuorial

Sirois Final Thesis – Comparison of the Fecal Microbiota of Horses Before and After Treatment for Parasitic Helminths: Massively Parallel Sequencing of the V4 Region of the 16S Ribosomal RNA Gene

 

Links to Bioinfomatics tools
-Got a question? Try asking at SeqAnswers – The Next Generation Community -It’s easy to join.  Another good one is BioStars which is more stats and bioinformatics based but lots of good info on experimental design with that end product in mind.

-Here is a quick guide to RNASeq tools (some specific to E. coli) –RNA-Seq data analysis tools overview

-DNAStar Lasergene 12- Available to use on BaseSpace but it has to be on the computer you are using in order to view results.  Google DNAStar to see all of it’s capabilities. We currently have a network license, which means it can be put on any Smith computer, but only 4 can be in use at one time, so be sure you quit when you are done. Lasergene 12 is loaded onto the AppleCart computers in the CMB – please be sure to sign out the AppleCart if you need a computer for this purpose.  Thanks

PATRIC – has a free suite of NGS analysis tools with storage space and tutorials for the most popular applications including de novo assembly.  Set up an account and check it out.

UniPro UGENE – has tuxedo suite set up for RNAseq and more. Easy to use.

Galaxy – Main page – hit “user” button and register and you are good to go.

DAVID – for functional annotation

MeV – Multi-Experiment Viewer – lots of stats and visualization tools – free download

– a quick guide to Making a heatmap of expression with MeV

Rockhopper – A free RNA Seq tool for bacteria studies offered by Wellesley College that assembles, aligns and gene counts your data and produces alignment, coverage, and expression results that can be viewed with IGV, as well as gene lists that can be further explored in databases such as BioCyc.

IGV at the Broad Institute – an Integrated Genomics Viewer free download and support

Qiime – Command line program for metagenomics analysis

Cytoscape – Network data integration, analysis, and visualization in a box – free download

MEGA – Molecular Evolutionary Genetic Analysis – free download

You are going to run into GO terms wherever you go.  Here is an article delineating where they come from and what they mean – Gene Ontology annotations what they mean and where they come from (1)

 

Links to Genome Browsers – these integrate a lot of tools for annotation and pathway placement.

BioCyc – database for everything else

Wormbase – database for nematodes

EcoCyc – database for E.coli K12

RegulonDB – Ecoli Wiki

GreenGenes – a 16S database and workbench

IF YOU WOULD LIKE TO ADD A LINK, PAPER OR PROTOCOL TO THIS SITE, OR IF THERE IS SOMETHING MORE YOU WOULD LIKE TO SEE ADDED TO THIS PAGE, PLEASE CONTACT LOUIE BIERWERT AT lbierwer@smith.edu  THANKS