RS_IsoSeq Protocol

Use this protocol to classify, cluster, and map cDNA sequences.

• Supports applications in transcriptomics.

• Enables the  functional characterization of transcripts and splice variants analyzed from the PacBio platform.

• Note: This protocol replaces the RS_cDNA_Mapping protocol in previous releases.

The protocol includes three main steps:

1. Classify: Extract reads of insert from PacBio movies; remove cDNA primers and poly-As; then classify the reads of insert into chimeric or non-chimeric, and full-length or non-full-length reads.

2. Cluster: Predict de novo consensus isoforms of transcripts from the classified reads using the ICE (Iteratively Clustering and Error Correction) algorithm.

3. Map: Using GMAP, align the classified reads and predicted consensus isoforms to the user-specific reference sequence.

Filtering Parameters  (IsoSeq Reads of Insert)

• Minimum Full Passes: The minimum number of full-length passes over the insert DNA for the read to be emitted.
• Minimum Predicted Accuracy: The minimum predicted accuracy (in %) of the reads of insert emitted.

Isoseq_classify Parameters (IsoSeq Classify v1):

• Minimum Sequence Length: The minimum length of the reads of insert to analyze.
• Customized Primers in FASTA: Path to a FASTA file containing customized primers used to detect primers in reads of inserts. (Optional)
• Full-Length Reads Do Not Require PolyA Tails: Specify whether or not full-length reads of inserts require PolyA tails.

• Maximum Number Of Paths Per Isoform or Read: The maximum number of GMAP paths to show for each isoform. If set to 0, outputs two paths if chimeras are detected; one path if chimeras are not detected. (This is the same as the GMAP --Npaths option.)

Isoseq_cluster Parameters  (IsoSeqCluster v1):

• Predict Consensus Isoforms Using The ICE Algorithm: Specify whether or not to predict consensus isoforms using the Iterative Clustering and Error Correction (ICE) algorithm.

• Parallel Tasks: The number of tasks to run in parallel while performing iterative clustering.

• Estimated CDNA Size: Specify the estimated cDNA sequence size - from under 1 kbp to over 3 kbp.

• Call Quiver to Polish Consensus Isoforms: Specify whether or not to use the Quiver algorithm to polish the resulting consensus isoforms.

• Minimum Quiver Accuracy to Classify an Isoform as HQ: The minimum Quiver accuracy needed to classify an isoform as high-quality.