Yes. The source code is identical for a given release, e.g. ICS v10.0 and SMRT Link v10.0. The only difference is how we generate the ccs binaries for SQIIe and SMRT Link; each binary is compiled for different environments, with varying degrees of hardware specializations and optimizations. ccs binaries for SQIIe are slightly faster than for SMRT Link, but all binaries from a given release yield identical results.
There are no plans to bifurcate. In fact, we dedicate a lot of energy into having all x86 binaries compile from the same source code and produce identical results.
Bioconda releases are only available as developer versions. They may contain features and bug fixes that have not been integrated in our release software yet.
SQIIe with kinetic information:
--all --all-kinetics --subread-fallback
Yes, those are mostly used to explicitly name auxilliary files. Everything explained:
/opt/pacbio/pa-ccs/current/bin/ccs \ # binary /data/pa/m64012_201202_224049.consensusreadset.xml \ # XML output name --all --all-kinetics --subread-fallback\ # all mode with kinetics --streamed \ # do not use a BAM file as input, but a BAM input stream --log-level INFO \ --suppress-reports \ # do not generate report or metric files per default --log-file /data/pa/m64012_201202_224049.ccs.log \ # log file name --bam /data/pa/m64012_201202_224049.reads.bam \ # explicit BAM file name --report-json /data/pa/m64012_201202_224049.ccs_reports.json \ # the ccs_reports file as JSON --report-file /data/pa/m64012_201202_224049.ccs_reports.txt \ # the ccs_reports file as human readable --metrics-json /data/pa/m64012_201202_224049.zmw_metrics.json.gz \ # metrics file name --hifi-summary-json /data/pa/m64012_201202_224049.hifi_summary.json \ # summary JSON file for hifi statistics --stderr-json-log # instrument-specific additional JSON logging
For HiFi runs, the main file is a
reads.bam file, prefixed by the movie name, and it is accompanied by two core auxilliary files:
The on-instrument ccs version and also SMRT Link ≥v10 run in the
--all mode by default. In this mode, ccs outputs one representative sequence per productive ZMW, irrespective of quality and passes. More information in the
--all mode FAQ.
Not when operating the instrument in CCS mode. See next question.
You can select either CLR or CCS reads coming off the instrument. If you want CLR and CCS reads, you can select HiFi generation on SMRT Link, which transfers CLR off instrument and runs CCS on your SMRT Link instance:
The design of SQIIe allows to have ccs running in parallel with the acquisition of the subsequent SMRT cell, and will not interfere or delay subsequent acquisitions. The hardware and software is configured to accommodate future increases in HiFi yield.
Performance of ccs on the instrument is roughly equivalent to running on a dedicated system with 384 logical cores. On-instrument processing includes parallel post-primary computation and result transfers up to 90% faster than for CLR runs.
There is no possibility to specify custom parameters. We are open to feedback what might be of general interest in future instrument software updates.
You may select to retain kinetic information in Run-Design:
Be aware that the output BAM files will more than 5x larger due to additional kinetics.
Can I generate a
reads.bam from a
subreads.bam file which was generated with older chemistry / instrument software (even from the Sequel System)?
reads.bam has been possible since ccs v5 and supported chemistries can be found here.
What is the file size of a HiFi dataset transferred from the instruments for one SMRT Cell on the SQIIe System?
We provide upper bounds and average file sizes how the BAM file size scales with HiFi yield in the BAM output FAQ. From this page, a typical 30 GBases HiFi yield run will result in a ~33 GByte
reads.bam file with default parameters and ~174 GB with HiFi kinetics included.
No, this currently not supported. Demultiplexing continues to be supported through SMRT Link or with lima on the command line.
reads.bam still be used in a CLR application? E.g. to make full use of all the data, not just the HiFi reads?
In SMRT Link and in general, only the HiFi reads are used from the
reads.bam. It should not be necessary to include other reads. Some pipelines do allow including CCS reads <Q20 (e.g. Iso-Seq). You are free to use the all reads from the
reads.bam files with your own tools.
Yes, the SMRT Analysis pipeline “Export Reads” in SMRT Link v10.0 or newer can export HiFi reads to BAM/FASTA/FASTQ format; when adjusting minimum CCS predicted accuracy, you can include CCS reads <Q20. On the command line, tools can be used to filter the BAM file for the read quality