******************** INTRODUCTION ******************** Last Updated: 01/18/2024 This README file describes the contents of this directory. This is a data release for Kinnex full-length RNA kit. The Kinnex full-length RNA libraries [1] were sequenced on the Sequel® II, IIe, and Revio Systems and processed using SMRT® Link v13.0 [2] or command-line/BioConda version [3]. To learn more about the Kinnex full-length RNA kit from PacBio for full-length RNA sequencing, read the application note [4]. ******************** SAMPLE ******************** ==================================== HG002 ==================================== Vendor – Coriell HG002 cells were purchased from Coriell and grown in RPMI1640 using Glutamax media with 16% FBS and 0.5% Penicillin-Streptomycin. RNA was isolated from 10x10^6 HG002 cells using Trizol reagent and Phasemaker tubes. RNA quality was assessed using a Bioanalyzer. ==================================== UHRR (Universal Human Reference RNA) ==================================== Vendor – Agilent Part No - 740000 UHRR total RNA was purchased from Agilent and directly used for cDNA generation. ==================================== Heart and Cerebellum samples ==================================== Collaborator – Seattle Children’s Research Institute (SCRI) Two heart samples from prenatal specimens with trisomy 21 were obtained from the Birth Defects Research Laboratory tissue repository. Total RNA was isolated from 50 mg of fresh frozen tissue using the Promega Maxwell Kit. Two heart samples from prenatal specimens were obtained from the Birth Defects Research Laboratory tissue repository. Total RNA was isolated from 50 mg of fresh frozen tissue using the Promega Maxwell RNA Extraction Kit. Three cerebellum samples were obtained from the Birth Defects Research Laboratory tissue repository. Total RNA was isolated from fresh frozen tissue sections or following laser capture microdissection using the Qiagen RNeasy Micro Kit as described in PMID:34140698. The collaborator has granted permission to release this dataset. ******************** METHODS ******************** Library Preparation: Procedure & Checklist - Preparing Kinnex libraries using Kinnex full-length RNA kit [1] Sequencing: Revio system with Revio polymerase kit and Revio sequencing plate. Sequel II and Sequel IIe system with Sequel II Binding kit 3.2 and Sequel II sequencing kit 2.0. Run time: Revio – 30 hr movie Sequel II/IIe – 30 hr movie Analysis: Read segmentation and Iso-Seq workflow ******************** FILE DESCRIPTION ******************** Each sample contains the following folders: ======================== 1-Sreads ======================== This directory contains segmented reads that were processed by Read segmentation to produce S-reads that represent the original cDNA molecules. segmented.bam contains S-reads that have the expected order of MAS adapters and is the file used in performing the subsequent analyses. 1-Sreads/ ├── segmented.bam ├── segmented.bam.pbi └── segmented.summary.json ======================== 2-FLNC ======================== This directory contains full-length, non-concatemer (FLNC) reads with the 5' and 3' cDNA primer as well as the polyA tail removed. FLNC reads are oriented from 5'->3' based on the asymmetry of the cDNA primers. 2-FLNC/ ├── flnc.bam ├── flnc.bam.pbi ├── flnc.filter_summary.json └── flnc.report.csv ======================== 3-ClusterMap ======================== This directory contains HQ cluster sequences by clustering FLNC reads at the isoform level de novo. The HQ cluster reads are then mapped using pbmm2 (minimap2 wrapper) to hg38. 3-ClusterMap ├── sample0.transcripts.bam (or clustered.bam) ├── sample0.transcripts.bam.pbi (or clustered.bam.pbi) ├── mapped.bam └── mapped.bam.bai ======================== 4-Collapse ======================== This directory contains the result of collapsing redundant isoforms after HQ cluster sequences were mapped to the genome. Each collapsed isoform has a unique ID `PB.X.Y` with an associated FLNC read count. 4-Collapse ├── collapsed_transcripts.fasta ├── collapsed_transcripts.flnc_count.txt ├── collapsed_transcripts.gff ├── collapsed_transcripts.group.txt └── collapsed_transcripts.read_stat.txt ======================== 5-Pigeon ======================== This directory lists the total set of unique transcripts as a result of mapping the clustered reads to the genome, collapsed into transcripts, and classified and filtered against Gencode using pigeon. Read about pigeon at [3]. The classification.txt and junctions.txt files are the output from pigeon showing the per-isoform and per-junction-per-isoform classification results against Gencode annotation. The GFF file shows the exonic structures of the transcript isoforms. The isoseq_saturation.txt file shows the rarefaction/saturation result of subsampling FLNC reads based on the post-filter pigeon result. 5-Pigeon/ ├── isoseq_classification.filtered.report.json ├── isoseq_classification.filtered_lite_classification.txt ├── isoseq_classification.filtered_lite_junctions.txt ├── isoseq_transcripts.sorted.filtered_lite.gff └── isoseq_saturation.txt ******************** REFERENCES ******************** [1] Procedure & Checklist - Preparing Kinnex libraries using Kinnex full-length RNA kit https://www.pacb.com/wp-content/uploads/Procedure-checklist-Preparing-Kinnex-libraries-using-the-Kinnex-full-length-RNA-kit.pdf [2] SMRT Link software https://pacb.com/software [3] isoseq.how https://isoseq.how/ [4] https://www.pacb.com/wp-content/uploads/Application-note-Kinnex-full-length-RNA-kit-for-isoform-sequencing.pdf Research use only. Not for use in diagnostic procedures. © 2024 Pacific Biosciences of California, Inc. (“PacBio”). All rights reserved. The data provided in these files and the information in this document are subject to change without notice. PacBio assumes no responsibility for any errors or omissions in the files or this document. Certain notices, terms, conditions and/or use restrictions may pertain to your use of PacBio products and/or third-party products. Refer to the applicable PacBio terms and conditions of sale and to the applicable license terms at pacb.com/license. Pacific Biosciences, the PacBio logo, PacBio, Circulomics, Omniome, SMRT, SMRTbell, Iso-Seq, Sequel, Nanobind, SBB, Revio, Onso, Apton, and Kinnex are trademarks of PacBio. All other trademarks are the sole property of their respective owners.