1.1 Post Run QC Analysis

Transcription

1 Post Run QC Analysis Post Run QC Analysis 1.1 Post Run QC Analysis Welcome to Pacific Biosciences' Post Run QC Analysis Overview. This training module will describe the workflow to assess initial key metrics related to the quality of data from a PacBio RS II analytical run.

2 1.2 Contents In this training module, we will present the following: A review of the basic definitions of Polymerase Reads and Reads of Insert Post run RS Dashboard metrics and how they can give an indication of the quality of data acquired We will also refer to additional resources available.

3 1.3 How to Use This Training Module This training module is intended to demonstrate how to interpret the primary analysis metrics displayed through the RS Dashboard following a sequencing run. Customers must reference PacBio protocols and guidelines for assessing run quality. Note: Deviations from the suggested acceptance criteria might still enable a run to be deemed successful.

4 1.4 Polymerase Reads & Reads of Insert Polymerase Reads & Reads of Insert

5 1.5 Polymerase Reads and Reads of Insert A SMRTbell template is a double-stranded DNA template capped by hairpin adapter loops at both ends. These hairpin adapters allow the SMRTbell template to effectively be considered topologically circular from the perspective of the polymerase. If the polymerase runs to the end of the insert, it will loop back to the opposite strand of the template molecule and continue sequencing until the movie acquisition period ends or the polymerase becomes deactivated. The term Polymerase Read refers to the contiguous sequence of nucleotides incorporated by the DNA polymerase during a sequencing reaction, for example while reading around a circular SMRTbell template. Polymerase Reads are most useful for quality-control monitoring of the instrument run. Polymerase Read metrics primarily reflect movie length and other run parameters rather than insert size distribution. Polymerase Reads are trimmed to include only the high-quality region; they include sequences from adapters; and can further include sequence from multiple passes around a circular template.

6 Each Polymerase Read is partitioned to form one or more subreads, which contain sequence from a single pass of a polymerase on a single strand of an insert within a SMRTbell template and no adapter sequences. The subreads contain the full set of quality values and kinetic measurements. Subreads are useful for applications like de novo assembly, resequencing, and base modification analysis. The Read of Insert represents the highest quality single sequence for an insert, regardless of the number of passes. A Circular Consensus Sequencing Read (CCS Read) is an example of a special case where at least two full-pass subreads are collected for an insert. Reads of Insert give the most accurate estimate of the length of the insert sequence loaded onto a SMRT Cell. For long templates, Reads of Insert may be the same as Polymerase Reads. If a SMRTbell template received only one-and-a-half subreads, that information will also be combined into a Read of Insert. Finally, a Read of Insert will also be produced in cases where the polymerase makes only 1 or fewer (i.e., incomplete) passes around the SMRTbell template.

7 1.6 RS Dashboard Metrics Assessment RS Dashboard Metrics Assessment

8 1.7 Post Run QC Key Metrics from RS Dashboard RS Dashboard is a web-based tool for monitoring runs and performance trends of a run or multiple runs. Accessing the RS Dashboard is very simple. Using web browsers Mozilla Firefox or Google Chrome, type < instrument> name /Metrics/RSDashboard This will take you to the RS Dashboard page where you can select a specific run or multiple runs. Included in individual run reports are summaries of the run parameters for each sample with statistics for each. The RS Dashboard also includes per-smrt Cell statistics to ensure that the resulting throughput and quality are as expected.

9 At the bottom of the report are charts for each SMRT Cell showing distributions of read lengths and accuracy, as well as graphs helpful for troubleshooting. 1.8 Post Run QC Key Metrics from RS Dashboard The key metrics to review after an analytical run are the following: Productivity & Loading Polymerase Reads: Length & Quality Reads of Insert: Length & Quality

10 1.9 RS Dashboard Metrics For information on RS Dashboard metrics, please move the cursor over the various buttons presented here. When you are finished, click on the next button located at the lower right corner of the screen to continue.

11 1.10 Productivity & Loading Productivity & Loading

12 1.11 Productivity Productivity': A measure of the number (yield) of reads generated from a ZMW. P=1 means that there is a polymerase read from that ZMW. P=0 means that a ZMW did not produce a read and is presumed to be lacking a polymerase. P=2 means other and the signal collected from the ZMW was not conducive to efficient base calling, possibly due to multiple templatepolymerase complexes bound in the ZMW and/or high background signal.

13 1.12 Loading Complexes into ZMWs Follows a Poisson Distribution This figure shows the relationship between overall ZMW loading and the number of singly loaded ZMWs. A critical step in the single-molecule-sequencing workflow is complex immobilization. This is a step where complexes are affixed to the bottom of ZMWs in preparation for sequencing. The process of immobilization is either passive using diffusion-based mechanisms, or MagBead-based mechanism. As a result, complex loading follows a Poisson distribution, which predicts that approximately 37% (or 55,000) of the ZMWs can be loaded with a single polymerase. Overloading will result in a reduction of the number of ZMWs loaded with single polymerases, and an increase in multiply loaded ZMWs.

14 1.13 Optimizing Loading Optimizing Loading Overloading may increase output of MB per SMRT Cell, but can increase multiply loaded ZMWs High Quality (HQ) region filtering can rescue some multiply loaded ZMWs, increasing the total number of reads per SMRT Cell Reads that have undergone HQ filtering have Shortened read lengths Lower accuracy compared to single-loaded ZMWs Evidence for increased chimeras These are less useful reads for de novo assembly Loading can be optimized through titration

15 1.14 Large Insert Example Let's look at a typical run for a large insert library using the following conditions: 20 kb size-selected library P5-C3 chemistry 180-minute movies MagBead loading Stage Start

16 1.15 Productivity & Loading Use the loading evaluation plots to evaluate loading performance. The left plot provides a sense of how much of the ZMW activity is low quality sequencing. Plotted are unfiltered vs. Polymerase Reads. Unfiltered reads are raw bases, which include low- and high-quality bases. Polymerase Reads are trimmed reads (low-quality bases are filtered) with bases in the high-quality region. A complete overlay of unfiltered and Polymerase Reads means most of the bases are high-quality with little trimming required. A large discrepancy between unfiltered and Polymerase Reads indicates there were noisy reads caused by multiply loaded zero mode waveguides that required significant trimming or filtering.

17 The plot to the right is also a good metric to measure overloading. A fraction of 1 means all called bases are in the high-quality region. As the low-fraction counts increase (as in the case of overloading), more and more reads have a low proportion of called bases located inside high-quality regions. In this example, there is very little discrepancy between the unfiltered and Polymerase Reads, which means loading is optimal. Additionally, most of the called bases are located in the HQ region. Together, this suggests that overloading is not an issue Polymerase Reads and Reads of Insert Polymerase Reads and Reads of Insert: Length and Quality

18 1.17 Polymerase Reads & Reads of Insert: Read Length In the example shown here, the average polymerase read lengths are approximately 9 kb. This is the average of the trimmed Polymerase Reads (HQ region only), which includes adapter sequences. The average read length for the Reads of Insert is approximately 8.3 kb. This is the contiguous, high-quality sequence with the adapter sequences removed. For large-insert SMRTbell templates, as in the case for 20-kb size-selected libraries, the read lengths of the Polymerase Reads and Reads of Insert should be close to being equal if loaded optimally (little to no trimming applied). Overloading and short-insert contamination will result in shorter Polymerase Reads and Reads of Insert.

19 For more information on this subject, please see the PacBio RS II brochure. The brochure shows representative data from a 20-kb size-selected E. coli library using a 180-minute movie utilizing P5-C3 chemistry where the average polymerase read lengths of approximately 8.5 kb have been demonstrated Polymerase Reads and Reads of Insert: Quality Polymerase Reads and Reads of Insert: Quality Polymerase read quality is a trained prediction of a read's mapped accuracy based on its pulse and base file characteristics. The read quality depends on the chemistry utilized. In this example, the read quality of the Polymerase Reads and Reads of Insert are the same.

20 1.19 Short Insert Example In the next example, a 1.6-kb amplicon library was created using P5-C3 chemistry, and was sequenced using 180-minute movies and MagBead loading.

21 1.20 Short Insert Example: Length As demonstrated in this example, the read lengths of the Reads of Insert closely match the expected insert size of the sample (1.6 kb). For short-insert libraries, the read lengths and read quality for Polymerase Reads and Reads of Insert differ. Depending on the movie lengths, the Polymerase Reads are longer than the Reads of Insert. This is because the Reads of Insert is the consensus sequence of all subreads from the polymerase read.

22 1.21 Short Insert Example: Quality For short-insert libraries, the read lengths of the Polymerase Reads and Reads of Insert differ. As demonstrated in this example, the read lengths of the Reads of Insert closely match the expected insert size of the sample (1.6 kb). The read quality will also be different with the Reads of Insert having a much higher read quality than the Polymerase Reads. This is achieved through the circular nature of the SMRTbell DNA template, which allows the polymerase to sequence the same base of the same DNA molecule multiple times. This is dependent on the insert size.

23 1.22 Additional Resources Additional Resources

24 1.23 Additional Resources Additional resources are available from the PacBio website.

25 1.24 Summary In summary, we have covered the post-run QC analysis process. In particular, we have described the basic definitions of Polymerase Reads and Reads of Insert. We demonstrated the assessment of the RS Dashboard metrics including: Productivity & Loading Polymerase Reads: Length & Quality, and Reads of Insert: Length & Quality. And finally, we have listed additional technical resources available from the PacBio portal.

26 1.25 Thank You Thank you for your participation. For more information, please contact your local PacBio Field Applications Scientist or PacBio account representative.