IsoSeq3

Scalable De Novo Isoform Discovery

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IsoSeq3 contains the newest tools to identify transcripts in PacBio single-molecule sequencing data. Starting in SMRT Link v6.0.0, those tools power the IsoSeq3 GUI-based analysis application. A composable workflow of existing tools and algorithms, combined with a new clustering technique, allows to process the ever-increasing yield of PacBio machines with similar performance to IsoSeq1 and IsoSeq2.

Availability

Latest version can be installed via bioconda package isoseq3.

Please refer to our official pbbioconda page for information on Installation, Support, License, Copyright, and Disclaimer.

Specific Version Documentation

• Version 3.1, SMRT Link (future version)
• Version 3.0, SMRT Link 6.0

What is new in version 3.1?

We outsourced the poly(A) tail removal and concatemer detection into a new tool called refine. Your custom primers.fasta is used in this step to detect concatemers.

FAQ

Why IsoSeq3 and not the established IsoSeq1 or IsoSeq2?

The ever-increasing throughput of the Sequel system gave rise to the need for a scalable software solution that can handle millions of CCS reads, while maintaining sensitivity and accuracy. Internal benchmarks have shown that IsoSeq3 is orders of magnitude faster than currently employed solutions and SQANTI attributes IsoSeq3 a higher number of perfectly annotated isoforms:

Additional benefit, single linux binary that requires no dependencies.

Why is the number of transcripts much lower with IsoSeq3?

Even though we also observe fewer polished transcripts with IsoSeq3, the overall quality is much higher. Most of the low-quality transcripts are lost in the demultiplexing step. Isoseq1/2 classify is too relaxed and is not filtering junk molecules to a satisfactory level. In fact, lima calls are spot on and effectively removes most molecules that are wrongly tagged, as in two 5' or two 3' primers. Only a proper 5' and 3' primer pair allows to identify a full-length transcript and its orientation.

I can't find the classify step

Starting with version 3.1, classify functionality has been split into two tools. Removal of (barcoded) primers is performed with PacBio's standard demultiplexing tool lima. Lima does not remove poly(A) tails, nor detects concatemers. For this, isoseq3 refine generates FLNC reads.

For version 3.0, poly(A) tail removal and concatemer detection is performed in isoseq3 cluster

My sample has poly(A) tails, how can I remove them?

Use --require-polya for isoseq3 refine. This filters for FL reads that have a poly(A) tail with at least 20 base pairs and removes identified tail.

How long will it take until my data has been processed?

There is no ETA feature. Depending on the sample type, whole transcriptome or targeted amplification, run time varies. The same number of reads from a whole transcriptome sample can finish clustering in minutes, whereas a single gene amplification of 10kb transcripts can take a couple of hours.

Which clustering algorithm is used?

In contrast to its predecessors, IsoSeq3 does not rely on NP-hard clique finding, but uses a hierarchical alignment strategy with O(N*log(N)). Recent advances in rapid alignment of long reads make this this approach feasible.

How many CCS reads are used for the unpolished cluster sequence representation?

Cluster uses up to 10 CCS reads to generate the unpolished cluster consensus.

How many subreads are used for polishing?

Polish uses up to 60 subreads to polish the cluster consensus.

When are two reads clustered?

IsoSeq3 deems two reads to stem from the same transcript, if they meet following criteria:

There is no upper limit on the number of gaps.

BAM tags explained

Following BAM tags are being used:

• ib Barcode summary: triplets delimited by semicolons, each triplet contains two barcode indices and the ZMW counts, delimited by comma. Example: 0,1,20;0,3,5
• im ZMW names associated with this isoform
• is Number of ZMWs associated with this isoform
• iz Maximum number of subreads used for polishing
• rq Predicted accuracy for polished isoform

Quality values are capped at 93.

What SMRTbell designs are possible?

PacBio supports three different SMRTbell designs for IsoSeq libraries. In all designs, transcripts are labelled with asymmetric primers, whereas a poly(A) tail is optional. Barcodes may be optionally added.

The binary does not work on my linux system!

Binaries require SSE4.1 CPU support; CPUs after 2008 (Penryn) include it.

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