Although the latest SMRTcell has been designed to shift the loading bias towards larger read lengths, when working with long insert libraries (10-20 kb), the preferential loading of smaller fragments often limits the potential of these libraries.
A solution to this is to remove small fragments from the libraries. We have evaluated the Blue Pippin (Sage Science, Inc., Beverly MA), an automated electrophoresis system that separates and collects DNA fragments based upon their size, for this purpose.
In order to measure the increase in subread length, long insert libraries were prepared with fragments larger than 4 kb or 7 kb isolated using the Blue Pippin and a 0.75% Agarose Gel Cassette (BLF7510) and compared to a library without Blue Pippin size selection. As shown below, the removal of smaller library fragments prior to sequencing increases the average length of the library fragments loaded into ZMWs on the SMRTcell.
In addition to longer subreads, there is also a boost to the amount of data generated per ZMW. As the fragment length increases, the percentage of SMRTbell adapter sequence decreases and the percentage of library insert increases. The graph below shows the average number of passed-filter bases per active ZMW versus the average fragment length of each library. Using Blue Pippin size selection, we have achieved yields of >500 M passed filter bases from individual SMRTcells.
Below are the sequencing and assembly results of four genomes sequenced from long-insert, Blue Pippin size-selected libraries. Using only PacBio long subread data, we were able to assemble complete microbial genomes for three of the four isolates. Even with only a single under-loaded and low-yield SMRTcell, the remaining isolate still resulted in a nearly complete genome assembly with 10 total contigs and >60% of the genome assembled in the largest contig.
Our PacBio throughput and read lengths have been improving steadily over the past year and may have just taken yet another big step forward. We upgraded our PacBio sequencer to RSII in mid-May and we are seeing significant increases in per-cell yield and improved read lengths with our longer libraries. The most notable change in the upgrade from RSI to RSII is the doubling of the number of simultaneously observable sequencing reactions on the SMRTcell, allowing throughput to be effectively doubled as well. Let’s take a look at some examples:
In this comparison of an 8kb Mycobacterium library that was run both before and after the upgrade, we see an almost 3x increase in total yield per-SMRTcell, while read lengths remain about the same.
Below is a comparison of per-SMRTcell stats from multiple libraries across multiple organisms, including both 8kb and 14kb libraries from Mycobacterium sp., Plasmodium falciparum, Saccharomyces cerevisiae and Candida albicans. Driven by the longer libraries, we see both dramatically higher yield and longer read lengths. On one recent 8 SMRTcell run of a 14kb library, we saw an average per-SMRTcell yield of 417 Mbp!
Here is a read length plot comparing the runs from the table above:
Although we are early in our use and optimization of the new PacBio RSII, we are encouraged by the increase in both yield and read length, and expect continued improvement in our PacBio data, subsequently improving data analysis and genome assembly.
We’ve spent some time recently testing a new way to assemble PacBio data called HGAP, which stands for “hierarchical genome assembly process”. Unlike previous assemblers of PacBio data that have relied on the use of either Illumina and/or PacBio CCS reads for error correction of PacBio long reads, HGAP uses multiple alignments of all reads to perform the corrections, potentially eliminating the need for other libraries and data types. The corrected reads are assembled with an overlap-layout consensus assembler (in this case Celera Assembler) to form contigs. More details about HGAP can be read found here: https://github.com/PacificBiosciences/DevNet/wiki/Hierarchical-Genome-Assembly-Process-%28HGAP%29
We have evaluated HGAP on several of our projects and compared it to our assembly of illumina-corrected Pacbio reads assembled with Celera Assembler. So far, the results have been very encouraging and we have seen significant improvement in many cases. The chart below shows several examples:
So the assemblies are more contiguous, but are the corrections good enough to generate accurate consensus sequence? In an attempt to verify the consensus accuracy of these HGAP assemblies for several Bordetella genomes, we aligned >240x coverage of 250bp Illumina MiSeq data to the HGAP-generated contigs and looked for discrepancies and SNPs using GATK. We found no cases of high-quality, passed-filter variants, which supports a highly accurate consensus sequence generated by the HGAP assembly. We continue to test and compare HGAP with other PacBio assembly methods but are encouraged by initial results.
Ken Dewar, McGill University, highlights how PacBio Circular Consensus Sequencing could be used to sequence ‘Rhino’viruses:
A new feature that was added with the recent PacBio upgrade is something called ‘Stage Start’. This allows for data collection to start earlier than it did previously. When this option is used, data collection begins immediately after the polymerase is activated, resulting in longer reads.
Below are the results from a quick test we performed. We sequenced two libraries with and without the ‘Stage Start’ feature turned on.
The libraries sequenced were about 8kb in length, and were sequenced using the Magbead Standard Seq v1 protocol. One 90-minute movie was taken of each SMRTcell. Standard Polymerase Binding and Sequencing kits were used (not the newer ‘XL’ version of the kits).
The PacBio was recently upgraded to version 1.3.3. With this upgrade comes the ability to use the XL versions of the DNA/Polymerase Binding and DNA Sequencing kits. These new kits should result in a longer average readlength (5000 bp) in comparison to the ~3000 bp average we get with the current C2 chemistry.
Using both new kits together does come at a cost. The data produced with the DNA Sequencing Kit XL 1.0 will be of a lower quality than with C2, and is recommended only when the data will be error corrected with shorter, more accurate reads.
For a boost in average read length without sacrificing quality of the reads, the DNA/Polymerase Binding Kit XL 1.0 can be used with the C2 sequencing chemistry rather than with the newer XL sequencing kit.
More details to follow…