PacBio Library Prep & Sequencing

Please also see the Introduction to PacBio Sequencing and General Information

PacBio RSII  and PacBio Sequel Sequencing: Services and Performance

We offer complete PacBio library prep, BluePippin size selection and sequencing services. The base calling and secondary analyses like Reads-Of-Insert extraction, HGAP assembly, and IsoSeq analyses of single or a small number of SMRT-cells are included in the service.  We will provide you the complete data set generated by the PacBio sequencer and SMRT-Portal analyses for download form our servers.  This includes FASTQ files of the filtered subreads as well as “.h5” raw data and intermediate files (bam files for the Sequel).  The Bioinformatics Core is offering in depth analyses of PacBio sequence data.  Samples have to be provided as isolated DNA by the customers.

The PacBio RSII Sequencer

The current chemistry generation for the RSII is called P6C4 and is used for all types of PacBio sequencing.  This chemistry has allowed to almost double the throughput per SMRT-cell as compared to its predecessor.
We are sequencing with both 4-hour (the default) and 6-hour movies. Please note that the latter only provides advantages for the highest quality DNA samples and libraries with insert sizes of 30 kb or longer.  Movies are processed in real time into trace files for each ZMW; base calls and associated quality scores are subsequently determined. These primary analysis files are passed through the secondary analysis pipeline to generate consensus sequence. Each SMRT-cell should generate between 35,000 and 70,000 good (single polymerase or “P1”) reads.  Since the precise quantification of the PacBio libraries before sequencing is not possible, the sequencing of the first SMRT-cell is mainly used to gather titration information and to QC the library. The yield of the first cell thus can be considerably lower than average. PacBio is continually improving read length and quality. The latest chemistry,  the P6-C4 chemistry, can produce average reads of up to over 14 kb and longest reads up to 70 kb (for 6 hour movies), given very high quality DNA samples. The official yield estimates provided by PacBio are 500 Mb to 1 Gb per SMRT-cell (after library titration).  We have seen average yields per SMRT-cell of up to 1.3 Gb (from very good DNA samples). Very long average read lengths are only possible after accurate size selection of the sequencing libraries (as smaller fragments will load preferentially). We offer the automated BluePippin size selection for enriching library fragments longer than 7 or 20 kb.  Please note that libraries with insert sizes greater than 20 kb may show reduced yields since large library molecules tend to load less efficiently.

The PacBio Sequel Sequencer

The Sequel is the second generation PacBio sequencer.  It generates 7x more reads per SMRT-cell compared to the RSII. The read lengths metrics are currently still lagging behind the RSII.  The Sequel chemistry is however undergoing rapid development and the read lengths are increasing.  The principles of the single molecule sequencing chemistry are unchanged. The sequence coverage and the sequencing errors do not show any detectable sequence-specific biases.  Pacific Biosciences has recently demonstrated a high quality genome assembly for Arabidopis based on the data of only two Sequel SMRT-cells.  The chemistry employed for the Arabidopsis project is now available (11/2016). Given high quality DNA samples and long insert libraries, mean polymerase-read-lengths of 10 kb can be expected and yields of around 5 Gb per SMRT-cell can be expected after titration.  Subreads can reach lengths of up to 35 kb.  Please note that currently several of the run metrics provided by the Sequel software are still unreliable. The important P1 percentage metric however can be relied on.  Accurate polymerase read length and subread lengths number will require alignment to a reference genome and running a “resequencing analysis”.   PacBio advises to tun 3 of 4 titration SMRT-cell to optimize the loading of the Sequel.  We expect that the number of required titration cells, similar to the RSII, can be reduced once all the run metrics are accurate and we have gained  more experience with this sequencer.

Sample Requirements, Sample Submission and Scheduling

PacBio library prep requires microgram DNA sample amounts. The recommended amount of input DNA further correlates with the desired read length – see these PacBio guidelines for SMRTbell libraries for details. Since the PacBio technology interrogates single molecules, any defect (e.g. a nick, an abasic site, a DNA adduct) can interfere with the sequencing process. Thus, the integrity and purity of the DNA sample is of utmost importance. The DNA quality and the DNA amount will determine which library insert sizes are feasible and how many SMRT-cells can be sequenced. The DNA samples should fulfill these criteria:

  • Minimal DNA purity: OD 260/280 should be 1.8-2.0; OD 260/230 should be >2.0
  • Has undergone a minimum of freeze-thaw cycles.
  • Has not been exposed to high temps (> 65°C for more than one hour can cause a detectable decrease in sequence quality).
  • Has not been exposed to pH extremes (< 6 or > 9).
  • Does not contain insoluble material.
  • Is RNA-free.
  • Has not been exposed to intercalating fluorescent dyes or ultraviolet radiation.
  • Does not contain chelating agents (e.g., EDTA), divalent metal cations (e.g., Mg2+), denaturants (e.g., guanidinium salts, phenol), or detergents (e.g., heme, humic acid, polyphenols).
  • Must be double-stranded. Single-stranded DNA cannot be converted into SMRTcell templates but can interfere with polymerase binding.

The sample requirements will vary strongly depending on genome size. Please contact us to discuss your project.  The table shows minimum requirements:

PacBio-requirements-012016-1024x167

One SMRT-seq library can provide sufficient material for the sequencing from as few as two to over 30 SMRT cells – depending on the amount and quality of the starting material and the desired size selection cutoffs. For example, for a 20 kb insert library we would ask for 20 ug high quality DNA to be able to use the BluePippin size selection​ (and submitting more DNA would be recommended). Lower sample amounts can be used with less stringent size selection, resulting however in reduced average read lengths. DNA samples are best dissolved in TE buffer at a high concentration (~ 100ng/ul or higher) and shipped on blue ice packs or wet ice.  Bacterial DNA samples are best isolated from cells in logarithmic growth phase.  Generally the sequencing of a single SMRT-cell combined accompanied by BluePippin size selection will be sufficient to assemble bacterial chromosomes as single scaffolds.
For difficult DNA samples, especially plant DNA samples with hard-to-remove contaminants (e.g. some polysaccharides), we recommend to carry out a high-salt/phenol/chloroform cleanup  (please note that this protocol often leads to a loss of 50% of the sample) or a purification with the BorealGenomics Aurora instrument (please inquire).
We will QC your sample by Pulsed-Field Gel Electrophoresis before library prep (BioRad CHEF, Pippin Pulse). A band (not a smear) of 50 kb or longer fragments indicates high integrity DNA samples desired for the generation of long insert size libraries. The final quality assessment of the DNA sample will however be the single molecule sequencing process itself (e.g the average read lengths). Bacterial DNA samples extracted using silica columns will be sheared by the spin columns to fragments of about 20 kb in size. Such bacterial samples tend to generate high quality data and are acceptable. This method is not recommended for eukaryotic samples.

Before submitting samples:

  • Please email us a picture of an agarose gel of the sample running also a marker with at least a 20 kb upper band (e.g. GeneRuler 1 kb Plus DNA Ladder or Lambda DNA/HindIII Digest Marker; suggested is a also a lane with undigested Lamdba phage DNA [48kb  e.g. NEB N3011S]). ​Please run the electrophoresis slowly (e.g. at 80V depending on setup).
  • Assess sample purity vie spectrophotometry  – the 260/280 ratio should be between 1.8 to 2.0 and the 260/230 ratio should be higher than 2.0. PacBio recommends MoBio PowerClean columns for sample cleanup or the high-salt/phenol/chloroform protocol mentioned above if necessary.
  • Please use fluorometric methods (e.g. Qubit) for DNA quantification if possible. Measure each sample at least 3 times and accept only reproducible measurements (HMW DNA is often not perfectly dissolved).  Spectrophotometry is not reliable for quantifications (especially if the DNA extraction protocol used CTAB).

The DNA samples used for making PacBio libraries must be handled with extreme care – if you need to ship your DNA to our facility, please consult the following PacBio guidelines for shipping and handling. More info and the submission form can be found on our Sample Submission & Scheduling page.   We must receive electronic and print copies of the submission form.

Prices

Costs for PacBio sequencing reflect the number of libraries and number of SMRT cells required. Our recharge rates can be viewed here.  The listed fees include all labor and reagents. BluePippin size selection is optional (and carries an additional fee), but is highly recommended. Please note our reduced high throughput (HT) recharge rates; these apply if 10 or more SMRT-cells are sequenced per sample or if 6 or more libraries are generated.