Sequencing Services

The Advanced Technology Genomics Core (ATGC) offers several sequencing services, including:

• Illumina next-generation sequencing
• Sanger-based DNA sequencing
• Sanger-based gene resequencing
• Single cell analysis

Learn more about each of these services below.

The ATGC provides a complete next generation sequencing service. Investigators provide the facility with genomic DNA, total RNA or ChIP DNA  (depending on the requested application) and the facility provides complete sample processing. 

NGS services include:

1.      Project consultation and budget planning with a facility representative and an MDACC faculty bioinformatician.

The ATGC provides free NGS project consultation.
Contact Erika Thompson ejthomps@mdanderson.org to make an appointment

*The ATGC does not guarantee output for investigator prepared libraries

**Please see the Price List for current External or GCC pricing.

NextSeq500-The Illumina NextSeq 500 System is the only desktop next-generation sequencing (NGS) system capable of sequencing a 30X human genome in a single run. Two flow cell formats and multiple reagent configurations enable data output from 20–120 Gb in a single run, providing flexibility across a broad range of applications. It has a simple workflow and quick run times that enable fast sequencing of exomes, transcriptomes, and whole genomes. The NextSeq 500 sequencer generates up to 400 million clusters passing filter (up to 120 Gb) in the High Output configuration and up to 130 million clusters passing filter (up to 40 Gb) in the Mid Output configuration.

*Please see the Price List for current External or GCC pricing.

MiSeq-the Illumina MiSeq is a low output sequencer capable of generating up to 15 Gb of data per instrument run.  It has the longest read-length in the Illumina line-up, generating up to 600 bases per 300bp paired-end run.  A variety of flow cells and read lengths provide flexibility on this single sample platform.

*Please see the Price List for current External or GCC pricing.

NGS Applications

Transcriptome Analysis

Transcriptome analysis may be quantitative (gene expression analysis) and/or qualitative (transcript discovery, splice variant identification, coding SNP validation, gene fusions).  The ATGC offers several options for transcriptome analysis.  The choice of sample preparation method is based on the sample quality, quantity and the investigator’s experimental objective.

Note: Strand specificity -Preserves strand information. Strand specificity can be used to identify antisense transcripts, determine the transcribed strand of non-coding RNAs and may help to demarcate the boundaries of overlapping genes.

Getting started

Project Consultation
The ATGC provides budget planning, technology consultations and project planning with an NGS specialist and an MD Anderson faculty bioinformatician. We strongly recommend that first time NGS service users and investigators with large-scale projects schedule a meeting before initiating a project. To schedule a consultation meeting please contact Erika Thompson ejthomps@mdanderson.org.

Sample Submission
All samples should be accompanied by a completed sample submission form. Sample submission requirements (minimum quantity and recommended sequence length) vary based on the service and sample type.

Data Analysis

Bioinformatics Faculty Collaborators

• Xiaoping Su, PhD.

Submit forms to
NGSsubmissions@mdanderson.org

The ATGC provides DNA Sequencing from single stranded or double stranded DNA, from purified plasmids, PCR products, and BACs. Sequencing is performed primarily on ABI 3730XL and 3730 DNA sequencers using Big Dye terminator cycle sequencing chemistry. The facility quantifies all samples, performs sequencing reactions, cleanup and capillary electrophoresis, analyzes the data and provides sequence as text files and chromatograms.

View our service pricing schedule for more information about DNA sequencing pricing.

Turnaround Time: 24-48 hours (weekdays)

Longer turnaround times may occur when our sample volume is very high, when special conditions are requested and when we experience instrument problems.

Guidelines for DNA submission

Sample submission requirements

Plasmid concentration must be 100 ng/µl. Submit 10 µl per reaction. Custom primers must be at 1 pmol/µl. Submit 10 µl per reaction. The DNA and primer must be submitted in 0.5-ml Eppendorf tubes with the sample name written on the sides and tops of the tubes. BAC DNA should be submitted at 500 ng/µl and primers at 25 pmol/µl. PCR products should be submitted at a minimum concentration of 20 ng/µl for products less than 1 kb. Products 1 kb or greater should be submitted at 30 ng/µl. Submit 10 µl per reaction

Quantitation of DNA

All DNA submitted to the facility needs to be accurately quantified. We recommend visual determination of DNA quality and quantity on an agarose gel using a quantitative DNA ladder. Alternatively DNA concentration can be determined fluorometrically. DNA concentrations determined using a spectrophotometer are often artificially high due to the presence of RNA, proteins, bacterial genomic DNA and other contaminants.

Note: Low DNA concentration is the most common cause of poor quality sequence and failed reactions. Too much DNA can, however, be as bad as too little. The presence of too much template results in top-heavy data (strong peaks at the beginning which fade rapidly), pull-up peaks (non-specific peaks that appear below the correct peak) and loss of peak resolution. In addition, it shortens the life of our capillaries.

Custom sequencing primer design guidelines

1. Thermal cycling conditions: Denaturing step heats the reaction mix to 96°C. The annealing step cools the reaction mix to 55°C, and the polymerization step extends at 60°C. Primers must have annealing temperatures of 56°C or higher.
2. Primer length should be at least 20- to 25-mers. GC content should be 50% or more.
3. Primers should be designed with a tightly binding 3' end.
4. When designing a primer, do not pick a region that is closer than 50 bases to the region of interest.
5. Primers for PCR reactions tend to work fine for automated sequencing.

Sequencing primers provided by ATGC

The facility currently provides the following primers free of charge:

• coreT3: CCT CAC TAA AGG GAA CAA AAG C
• coreT7: TAA TAC GAC TCA CTA TAG GGC GA
• coreT30: ATT AAC CCT CAC TAA AGG GA
• coreT70: TAA TAC GAC TCA CTA TAG GG
• coreM13F: GTA AAA CGA CGG CCA G
• coreM13R: TCA CAC AGG AAA CAG CTA TGA C
• coreSp6: GAT TTA GGT GAC ACT ATA G
• coreBluescriptKS: TCG AGG TCG ACG GTA TC
• coreBluescriptSK: CGC TCT AGA ACT AGT GGA TC
• coreBGHRev: TAG AAG GCA CAG TCG AGG
• corePCMVFor: CGC AAA TGG GCG GTA GGC GTG
• corepGEX3: CCG GGA GCT GCA TGT GTC AGA GG
• corepGEX5: GGG CTG GCA AGC CAC GTT TGG TG
• coreT7Ter: GCT AGT TAT TGC TCA GCG G
• corePolyA: AAA AAA AAA AAA AAA AAA AAA AAA T
• corePolyT: TTT TTT TTT TTT TTT TTT TTT TTT A
• coreU6: GAG GGC CTA TTT CCC ATG ATT

Redo policy

The facility will repeat a sample at no cost to the investigator if there is an instrument failure, or if the quality of sequence is compromised due to an error in the facility. If the investigator recommends a sample be repeated, the same DNA and primer will be used to repeat the sequencing reaction. If the reaction fails a second time, the investigator will be charged for the repeated reaction. If an incorrect primer has been requested for sequencing and as a result no sequence data is produced by the sequencing reaction, the cost will be borne by the investigator.

The facility will assist investigators in designing primers to difficult regions. Please contact Erika Thompson if you require this service

Frequently asked questions

View answers to frequently asked questions such as: Why did my template not sequence? Why should I resuspend in water? Does the host strain matter? Does it matter which media I use to grow my cells? Why did my sequence stop short?

Accessing results

Downloading sequences

*Note: The text sequence provides you with unedited, raw sequence data. Please view the chromatograms to correct minor errors made by the base-calling software. The Sequencing and Microarray Facility provides a server for the rapid dispersal of data to the principal investigators. The results remain on the server for 30 days, after which files will automatically be deleted. Investigators should copy all data to their drives.

AppleShare


When using AppleTalk, the server will only allow 20 users to log on at a time. The server has been set up to allow 15 minutes per user log on to download files. If your workstation logs on automatically at start up you will be bumped off the system after 15 minutes.
(If you do not have a folder online, please contact the ATGC and we will have a folder created for you.)

New Directions to Access Your Online Sequencing Results

PC users:

• Click "Start"
• Click "Run"
• Type in “\\mymdafiles\seq”
• Click "OK"
• A dialog box will appear. Type in your "username" and "password".

Macintosh users:

• Click on "Go" (located at top of screen)
• Click on "Connect to Server"
• Type in “smb://mymdafiles/seq”

Both PC and Mac users will use the same username and password that you use to access your MD Anderson account through Entourage and/or Outlook.

Viewing chromatograms (free downloadable software)

PC users:
Chromas Software: http://technelysium.com.au/wp/chromas

Mac Users:
4peaks Software: http://downloads.nucleobytes.com/4peaks

Sanger-based gene resequencing enables gene mutation detection by evaluating an entire gene or individual SNPs in a single experiment. The ATGC performs this assay using custom designed primers. View the genes available for this service.

View the service pricing schedule for more information about gene resequencing pricing.

Service highlights

• Customization available
• New assay design free of charge
• Gold standard for next-generation sequencing validation
  

Instrumentation

Quantification is performed on a Qubit fluorometer. Sequencing is performed on a 3730XL DNA Analyzer (Thermo) and comparative analysis uses SeqScape software (Thermo).

Sample workflow

1. Quantification and normalization of samples
2. PCR amplification and purification
3. Sanger sequencing in both directions (where possible)
4. Comparative alignment to the reference sequence
5. Re-analysis (if needed)
6. Report of all mutations found and all sequences generated sent to customer

Sample submission

Send completed submission form to D.J. Doss or submit hard copy with samples. Samples may be submitted in 1.5 or 0.5 microcentrifuge tubes with the name clearly written on the top of the tube. The amount of gDNA needed is dependent on the gene requested, please contact D.J. Doss for more information.

Additional information/documents

• Gene List
• Pricing
• Submission Form - email D.J. Doss for current form(s)

Scheduling sample drop-off

Contact D.J. Doss to schedule sample drop-off and to determine the amount of gDNA needed.
For new assay design please contact D.J. Doss or Erika Thompson.

Contact information

D.J. Doss
Email: djdoss@mdanderson.org

Erika Thompson
Email: ejthomps@mdanderson.org

The ATGC offers single cell analysis using the 10X Genomics Chromium and Mission Bio Tapestri platforms

10X Chromium Single Cell Analysis

Submitting samples for 10X single cell processing

Project Consultation - We strongly recommend a consultation meeting before starting your first 10X Genomics project. Consultations are free for investigators utilizing the ATGC's single cell service. To request a meeting please contact David Pollock (dpollock@mdanderson.org) or Erika Thompson (ejthomps@mdanderson.org).

Scheduling Your Experiment - The single cell service is by appointment only. You must have an appointment prior to submitting samples. Submission appointments should be made a minimum of one week in advance of your anticipated submission date. This prevents scheduling conflicts and ensures the appropriate reagents are available and at the correct temperatures for immediate use (having samples sit while we bring reagents to temperature may negatively impact data). We will make effort to accommodate appointments made 24-48 hours before submission but we cannot guarantee availability.

To schedule a submission appointment, please contact David Pollock (dpollock@mdanderson.org).

Sample Submission – To submit samples please complete a 10X Genomics Single Cell Service Request Form and email the completed form (with an active account number and the appropriate signature) to David Pollock (see email above).  For external investigators, an additional external billing form with PO# and PI signature authorization is required in addition to the service request form.

Single Cell Applications 

3' scRNAseq Gene expression profiling

Applications Highlights

• Gene expression profiling.
• Capture up to 10,000 cells per well of partitioning chip

Workflow - Single cells, RT priming-beads with specific barcode and universal molecular identifier (UMI), as well as lysis and RT reagents are partitioned into oil droplets where the beads are dissolved and cells are lysed for reverse transcription to take place. The droplets are then broken and the pooled single-stranded, barcoded cDNA are amplified and fragmented for library preparation. During the library preparation process, appropriate sequence primer sites and adapters are added so that the final product contains the 10X Barcode, UMI, the appropriately sized cDNA insert and Illumina adapters with  P5 and P7 primer sequences for sequencing on an Illumina Sequencer (typically the NovaSeq6000 or NextSeq500).

5’ scRNAseq gene expression with immune profiling

Application highlights

• Gene expression profiling with the added ability to immune profile T cell TCR and B cell Ig for both human and mouse samples.
• Capture up to 10,000 cells per well of partitioning chip.

Workflow - The workflow for 5’ gene expression is similar to that of 3’ scRNAseq  with the additional ability to enrich for the V(D)J segments expressed in T cells or B cells after cDNA amplification is completed.   This provides the capability of running gene expression profiling, TCR profiling, and Ig profiling from the same cell suspension. 

scATACseq

Application highlights      

• Determination of single cell open chromatin regions for understanding epigenetic and regulatory variation across the genome.
• Capture up to 10,000 nuclei per well of partitioning chip. 

Workflow - The single cell ATAC assay is used to assess the accessibility of chromatin on a single cell level. In the first step of this workflow, single nuclei are isolated from the single cell suspensions. Next, the nuclei generated in the first step undergo a transposase enzymatic reaction where accessible DNA regions are fragmented and tagged with sequencing adaptors. The transposed nuclei are then partitioned into GEMs where each nuclei is individually barcoded and made ready for library preparation.

Multiome (scATACseq and 3’ scRNAseq)

Application highlights      

• Determination of single cell open chromatin regions for understanding epigenetic and regulatory variation across the genome.
• Gene expression profiling.
• Has the added benefit of scATACseq and scRNAseq obtained from the same cell.
• Capture up to 10,000 nuclei per well of partitioning chip. 

Workflow – This application has a similar workflow as the scATAC seq application.  In the first step of this workflow, single nuclei are isolated from the single cell suspensions. Next, the nuclei generated in the first step undergo a transposase enzymatic reaction where accessible DNA regions are fragmented and tagged with sequencing adaptors. The transposed nuclei are then partitioned into GEMs where each nuclear material in each nuclei is barcoded.    The resulting barcoded nucleic acid content is then amplified.  The resultant amplified product is used for both the scATAC library generation as well as the cDNA amplification and library preparation for the 3’ scRNAseq component.

Sample requirements

Sample Type - Samples should be submitted as single cell suspensions in 1.5 mL microfuge tubes.   Cell dissociation from tissue and removal of significant cell debris via filtration (e.g. 40 um Flowmi tip cell strainer) should be performed (by the investigator) prior to submission.

Formalin-Fixed cells are not suitable for use in any 10X Genomics protocol.

Cell enrichment - by Flow cytometry, magnetic bead positive or negative cell selection, etc.. should be performed by the submitting investigator prior to sample submission.

Viability - Optimal viability is >90% however viabilities of 70-90% are acceptable.  Viable cell suspensions can be cryopreserved (viability >90% prior to cryopreservation) and then submitted at a later time. Please note that the viability of previously cryopreserved samples can vary greatly.  A Low viability may negatively impact the efficiency of cell capture as well as the sequencing data.

Buffer - Single cell suspensions for 3’ or 5’ scRNAseq services can be submitted in either 1X PBS (with ≤ 0.04% BSA-recommended by 10X Genomics-ideal buffer) or, for more sensitive cells, culture media.  Please note, that additives in media can have an effect on capture efficiency.  At the minimum, EDTA/EGTA should be left out of the media since it will inhibit downstream enzymatic steps.   If bringing samples in PBS, cells should have been freshly harvested (minimal transit time before submission); otherwise, cells in media may be better depending duration of the cells outside of the system or on the sensitivity of your cell suspension in a non-media environment.  Fetal bovine serum is permitted in either PBS or media but should not contain more than 10% fetal bovine serum (5% or less preferred).

scATACseq cell suspensions should be submitted in 1X PBS.  We do not recommend media for either application.

Free Nucleic Acids- If samples are suspected of containing contaminating free nucleic acids (from dead, dying, or lysed cells), we recommend that you remove them from the cell suspension by low-speed centrifugation (300-500XG for 5 minutes at 4 degrees C) of the cells along with 1-2 washes with fresh PBS/media.   The presence of free nucleic acids negatively impacts the efficiency of cell capture as well as the sequencing data..

Cell concentration

3’ or 5’ scRNAseq -The optimal cell suspension concentration is 1000-2000 cells/uL  (100,000 total cells in 100 uL buffer).   We can, however, run lower cell quantities (we have successfully run cells < 10,000).  For low cell quantities the volume of cells required is a maximum of 10-30 uL.

scATACseq,- The cell input for this application is a minimum of 100,000-200,000 cells in 100-200 uL of PBS.    Lower cell inputs are permitted in the range of 2,000-40,000 total cells in a volume of 100 uL of PBS.

Multiome (scATAC and scRNAseq)- The cell input for this application is a minimum of 100,000-200,000 cells in 100-200 uL of PBS.   

Sequencing Requirements

10X Genomics has minimum reads/cell recommendations for each application.  These are very broad guidelines and may not meet the needs of your specific project. We strongly recommend that you consult your biostatistical collaborator to determine the number of cells and reads per cell required to meet your specific experimental objectives. 

The table below outlines the ATGC’s  general recommendations for the number of read pairs/cell for each application.   The table is meant only as a guide for sequencing coverage.

Single cell sequencing is typically performed on the Illumina NovaSeq6000 or NextSeq500 sequencers.   Both sequencers allow flexibility by providing various flow cell format options to meet your sequencing requirements.   The choice of sequencer and flow cell format for single cell experiments depends on the number of samples submitted and the target cell captures requested for each sample.  For high sample volume or complex sample library pools, we will prescreen for appropriate sample sequencing distribution using the iSeq100 prior to sequencing on higher density flow cells.

Data Output

The ATGC only provides raw data generated from our service. Base Call files (bcl) are converted to fastq files using specific software from 10X Genomics. Modified fastq files are used for QC metrics (html files) and demultiplexing of the sequencing data using 10X Genomics Application Pipeline software. Analysis files are created using 10X Genomics Pipeline software which can be viewed using freeware analysis software which is available for download from 10X Genomics.

Please see single cell service and sequencing pricing on the ATGC Price List. ATGC Price List.

Targeted Single Cell Multi-Omics

The Mission Bio Tapestri  Platform analyzes genotype and phenotype from the same single cells.

Application highlights

• Simultaneous detection of SNVs, CNVs, and protein at the single-cell level
• SNV/indel detection across 100’s of loci
• Gene and chromosome level CNV detection
• Sensitivity for rare clones- down to 0.1%
• Capture 5k-10K cells for DNA only, 2k-10K for protein
• Minimum panel: 20 amplicons, maximum 1K amplicons
• Off the shelf or custom panels
  
• Human and mouse panels

The panels are configured to run SNV alone, SNV in combination with CNV, or a triple combination of SNV, CNV, and protein detection.     

Mission Bio provides target panels for hematologic tumors, solid tumors, or the capability of custom panel creation.   The ability of providing custom panels also allows for the ability to determine the status of genome editing (e.g. CRISPR/TALEN) from engineered DNA sequence edits. Specific information for each panel can be located on Mission Bio’s website link: https://missionbio.com/panels 

Technology Overview - 

The Tapestri microfluidics system is used to partition single cells (up to 10,000) and a protease enzyme mix into oil droplets where the cells are lysed.  The resultant cell lysate partitions are then partitioned a second time into droplets that include barcoded beads, primers, and reagents for generating uniquely barcoded amplicons.  Following amplification, the barcoded-DNA amplicons are used to generate Illumina compatible libraries.  

During library preparation Illumina P5 and P7 sequencing adapters are added to the amplicons to facilitate sequencing on the Illumina platform.   Following sequencing, the fastq files generated are analyzed using Mission Bio’s analysis software.  

Sample Requirements

Sample Type - Samples should be submitted as single cell suspensions in 1.5 mL microfuge tubes.   Cell dissociation from tissue and removal of significant cell debris via filtration or cell washes should be performed by the investigator prior to submission.   If samples are suspected of containing contaminating free nucleic acids (from dead, dying, or lysed cells), we recommend that you remove them from the cell suspension by low-speed centrifugation (300-500XG for 5 minutes at 4 degrees C) of the cells along with 1-2 washes with fresh buffer.   The presence of free nucleic acids could negatively impact the efficiency of cell capture as well as the sequencing data results. 

Formalin-Fixed cells are not suitable for use in this application. 

This technology is not currently designed for RNA transcriptome analysis.   Please see 10X Genomics services for scRNAseq services. 

Cell enrichment - Flow cytometry, magnetic bead positive or negative cell selection, etc.. should be performed by the submitting investigator prior to sample submission.

Viability - Optimal viability is >90%; however, viabilities >70% are acceptable.  Viable cell suspensions can be cryopreserved (viability >90% prior to cryopreservation) and then submitted at a later time. Please note that the viability of previously cryopreserved samples can vary greatly.  A Low viability may negatively impact the efficiency of cell capture as well as the sequencing data.   Cell nuclei protocols can be utilized for cells that have previously been frozen. 

Cell suspension buffer - 1x DPBS w/o Ca2+/Mg2+.   Note:  This protocol has not been validated for cells submitted in media.    

Cell concentration:

Optimal: 400K total cells in a volume of 100 uL of 1x DPBS w/o Ca2+/Mg2+.  (4000 cells/uL) Minimum: 200K total cells in a volume of 50 uL. 

Minimum: 200K total cells in a volume of 50 uL. 

When possible we recommended providing a higher concentration to allow for possible differences between our facility and another laboratory’s concentration measurements.     

Sample QC 

Cell concentration and viability are determined using trypan blue exclusion detection and read on an automated counter.   Any samples not meeting our viability requirements will also be evaluated manually under an inverted microscope where an estimate will be provided.  The investigator will be able to decide at that time if they wish to proceed with processing. 

Sequencing Requirements

Sequencing requirements vary based on the panel selected.

AML panel: ~75M read pairs

Myeloid panel: ~188M read pairs

CLL panel: ~165M read pairs

THP panel: 146M read pairs

Supported sequencers that are available are MiSeq, NextSeq500, and NovaSeq6000. Most panels require 150nt paired end sequencing with 8nt dual indexes. 

Data Output - Raw data fastq files will be used for data analysis.  Our bioinformatics will perform the initial analysis, which includes the read alignment, variant calling, QC metrics and loom file generation.   The Loom files generated can be utilized in Mission Bio’s Tapestri Insights analysis software which will enable further data analysis, variant filtering, and data visualization.  Tapestri Insights software can be downloaded from Mission Bio’s website: www.missionbio.com

Getting Started

Project Consultation - We strongly recommend a consultation meeting before starting your first Tapestri project. Consultations are free for investigators utilizing the ATGC's single cell service. To request a meeting please contact David Pollock (dpollock@mdanderson.org) or Erika Thompson (ejthomps@mdanderson.org).

Scheduling Your Experiment -

The single cell service is by appointment only. You must have an appointment prior to submitting samples. Submission appointments should be made a minimum of one week in advance of your anticipated submission date. This prevents scheduling conflicts and ensures the appropriate reagents are available and at the correct temperatures for immediate use (having samples sit while we bring reagents to temperature may negatively impact data). We will make effort to accommodate appointments made 24-48 hours before submission but we cannot guarantee availability. 

To schedule a submission appointment, please contact David Pollock (dpollock@mdanderson.org). 

Sample Submission – To submit samples please complete a Mission Bio Tapestri Single Cell DNA Service Request Form and email the completed form (with an active account number and the appropriate signature) to David Pollock (see email above).  In addition external submissions require a completed external billing form and an e-copy of the purchase order. 

The ATGC now offers the following data analysis service to support our complete (library preparation and sequencing) NGS service:

            Notes:

1. Analysis is available for ATGC full NGS service (library preparation and sequencing) only.
2. Projects with >50 samples require a mandatory consultation prior to analysis.

Download the NGS Data Analysis submission form.