Next-Generation Sequencing

Next-Generation Sequencing

Library preparation, sequencing, bioinformatics.

RNAseq, ChIPseq, ATACseq, CRISPR screening, or something new!

Specific NGS Applications

RNAseq Considerations

Gene Level Differential Expression Testing and Pathway Analysis

The most commonly used, cost effective and streamlined RNAseq service in our facility is mRNAseq (polyA enriched) library prep, single-end 75bp sequencing on NextSeq of 12-16 pooled barcoded samples, and VIPER analysis. These RNAseq data are adequate for gene level differential expression profiling and pathway analysis and equates to 25-30M reads/sample. We recommend at least 3 biological replicates per condition.

Alternative Library Prep Options (see Library Prep tab below for more details)

IF you have low amount of RNA (<250ng) or low cell count from sorting, your samples may require low input RNAseq library preparations using Clontech SmartSeq v4 chemistry.
IF your RNA is degraded OR you are interested in non-polyA lncRNAs, you should consider our rRNA depletion totalRNAseq library prep.
IF you are interested in small RNAs, you should consider our small RNA sequencing service as small RNAs such as miRNAs are not reliably captured and quantified in standard RNAseq library prep methods.

Sequencing: Single-end vs. Paired-end (see Sequencing tab below for more details)

IF you are interested in transcript level analysis (isoforms), detecting gene fusions, calling variants, detecting viral transcripts, or other specialized analysis, you should consider paired-end 75bp sequencing.
IF NOT, single-end 75bp is fine.


RNAseq Workflow

  • Submit purified DNase treated total RNA – either in the -80C freezer in the “Fish Room” or directly to our facility on the 4th floor of DFCI Fenway at 21-27 Burlington Ave.
  • MBCF will QC samples shortly after receipt (~2 days). In the event that your samples do not meet our quality or quanitity requirements, you will be notified and given the opportunity to replace any failed or questionable samples before moving forward. We will also provide relevant recommendations based on our experience.
  • Library preparation is typically completed within 2 weeks (sample volume dependent).
  • Completed libraries are individually measured by Qubit, TapeStation and Kapa qPCR quant assay, pooled accordingly, and sequenced.
  • Raw data are distributed internally through server share folders setup by DFCI Research Computing (if you don’t have server access yet, request it). Data are distributed to external investigators by web server, ftp, or an old fashioned external hard drive.
We DO NOT perform chromatin IP in our facility, but we can make ChIPseq libraries from a small amount of DNA.

DNA QC: bioanalyzer
Library Prep Method: Rubicon Genomics Thruplex DNAseq
Sequencing: MiSeq (1 or 2 samples), NextSeq (10-20 samples)
Analysis pipeline: ChiLin

Screening

Library preparation: PCR prep performed by investigator. Here is an efficient PCR library prep protocol from the Broad Institute that works well. We have developed software capable of demultiplexing these staggered primer barcode designs.
QC: MBCF will characterize library by Qubit fluorometer, Agilent TapeStation, and Kapa Quant qPCR assay
Sequencing: For genome-wide screens of many samples, NextSeq SE75bp is generally recommended. For fewer samples and/or fewer gRNA targets, MiSeq SE75bp is often more appropriate.
Analysis: The MBCF currently supports a rudimentary pipeline that will demultiplex libraries, map the gRNAs with bowtie to a gRNA reference list (either standard GeCKO LibA/LibB or custom user defined target list), and return a count matrix.

Coming Soon
Targeted CRISPR editing indel characterization service

For more comprehensive information about CRISPR technology:
https://www.addgene.org/crispr/guide/

General NGS Services

One of the most exciting and fun aspects of NGS is the wide variety of library prep and sequencing applications. Here are a few that we do regularly. If you want to try something else, let us know! We want to try it too.

RNA

Illumina stranded mRNAseq

Description: The most commonly used RNAseq library prep method (in our facility). This preparation method uses oligo dT beads to enrich for mature polyA(+) transcripts.

Sample Requirements: >500ng high quality (RIN > 7) purified DNase treated total RNA.

Considerations: Does not detect transcripts that are not polyadenylated and although miRNA are sometimes detected the quantification will not be reliable

Illumina stranded totalRNAseq

Description: rRNA depletion followed by RT priming with random hexamers, cDNA synthesis, and adapter ligation. Compatible with degraded samples.

Sample Requirements: >500ng purified DNase treated total RNA

Considerations: Requires greater sequencing depth per samples to achieve same coverage of coding content as mRNAseq libraries as ~40% of reads will map to introns.

Clontech SMART-Seq v4

Description: Low input RNAseq method that uses oligo dT RT priming and template switching to generate full length cDNA from very limited amounts of RNA (10pg-10ng).

Sample Requirements: <10ng high quality (RIN >7) total RNA.  Protocol modifications possible to work from cell lysate.

Considerations: Appropriate for very low cell counts down to single cell. Some technical variation may occur with <500pg of input. After full length cDNA synthesis, Illumina libraries can be constructed by shearing the DNA and ligating adapters or by the Nextera transposes based prep. Shear+ligate is preferable, but Nextera is more cost effective for large numbers of samples such as sorted single cells.

Small RNA

Description: RNAseq library preparation targeting the small RNA fraction that is purified away during standard RNA preps such as mRNAseq and totalRNAseq.

Sample Requirements: 1ng-1ug purified totalRNA or enriched small RNA. If using a total RNA extraction method be sure that it preserves the small RNA fraction.

 

DNA

Adapter Ligation

Rubicon Thruplex DNAseq is an efficient adapter ligation method that is well suited for low input libraries (<5ng), but also works well with 50ng input.

Sample Requirements: 1ng-50ng, fragmented DNA (typically Covaris sonication)

Applications: General Use, ChIPseq

Illumina Nextera

Nextera is a fast library prep method that requires only a small amount of DNA -as little as 1ng of input. This method uses a transposase to simultaneously fragment and tag DNA with Illumina adapters (“Tagmentation”) followed by a limited cycle PCR reaction to incorporate sequencing primers and indices.

NexteraXT: 1ng input DNA -smaller yield (ideal for multiplexing), 12 PCR cycles.

Applications: Small genomes, large amplicons, ATACseq

Illumina Sequencing by Synthesis

Need a refresher? This 5 min. video from Illumina might help.

Sequencing reagents are priced by the number of “cycles”. Sequencing by synthesis means that 1 chemistry cycle = 1bp.  Cycles are typically divided symmetrically. For example, a 150cycle reagent kit is typically used to perform paired-end 75bp sequencing. However, cycles can be allocated asymmetrically (upon request) for customized read lengths such as 37bp X 51bp.

NextSeq

The Illumina NextSeq500 is a fast high throughput sequencer that is capable of generating data from >400M clusters in 12-30hrs.

Mid Output (MO) = 130M clusters (reads or read pairs)

High Output (HO) = 400M clusters (reads or read pairs)

Pricing

Reagent Kit DFCI External
75cycle (HO) $2,200 $2,860
150cycle (MO)  $2,200  $2,860
150cycle (HO) $4,000 $5,200
300cycle (MO)  $2,500  $3,250
300cycle (HO) $6,500 $7,250

MiSeq

The Illumina MiSeq is a single-lane “low-throughput” sequencer.

Version 2 chemistry (v2): 12-15M clusters (reads or read pairs)

Version 3 chemistry (v3): 20-25M clusters (reads or read pairs)

Pricing

Reagent Kit DFCI External
50cycle (v2) $850 $950
150cycle(v3) $1,200 $1,300
300cycle(v2) $1,200 $1,300
500cycle(v2) $1,600 $1,700
600cycle(v3) suspended suspended

Visualization Pipeline for RNAseq (VIPER)

In collaboration with the Center for Functional Cancer Epigenetics (CFCE) at Dana-Farber Cancer Institute, the MBCF has developed a Visualization Pipeline for RNAseq (Viper) analysis tool.

viper_overview

Standard output includes:
-Aligned sorted.bam files
-Raw gene count and normalized FPKM matrices
-DESeq2 differential expression testing results
-Graphical Summary Report

ChIPseq analysis (ChiLin)

This pipeline was developed by the Center for Functional Cancer Epigenetics at Dana-Farber Cancer Institute. Reads are aligned with bowtie or BWA if paired-end followed by MACS for peak calling.

Output Includes:
-bigwig files for visualization
-peak list
-ChIPseq QC summary .pdf

CRISPR screening

This pipeline is currently in development.

– Demultiplex staggered inline barcodes (if using Broad protocol)
– Align reads with bowtie to reference gRNA sequences (if not GeCKO LibA or LibB, user must provide)
– Generation of count matrix