The Iso-Seq library was prepared according to the Isoform Sequencing protocol (Iso-Seq) using the Clontech SMARTer PCR cDNA Synthesis Kit and the BluePippin Size Selection System protocol as described by Pacific Biosciences (PN 100–092–800-03). Sequence data were processed using the SMRTlink 5.0 software. The CCSs from subread BAM files (parameters: min length = 200, max drop fraction = 0.8, no polish TRUE, min z-score = − 9999, min passes = 1, min predicted accuracy = 0.8, max length = 18,000) were classified into full length and non-full length reads using pbclassify.py script, ignore poly-A false, min Seq Length 200. Non-full length and full-length reads were then got into the clustering step, which does isoform-level clustering (ICE), followed by final arrow polishing. Additional nucleotide errors in consensus reads were corrected using the Illumina RNAseq data with the software LoRDEC [68 (link)].
Lordec
Manufactured by Illumina
LoRDEC is a software tool designed for error correction of long reads obtained from next-generation sequencing platforms. It utilizes a combination of short and long reads to identify and correct errors in the long reads, improving their accuracy and quality for downstream applications.
Automatically generated - may contain errors
5 protocols using lordec
1
Isoform Sequencing Workflow
2
Single-Molecule Long-Read Transcriptome Analysis
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Sequence data were processed using the SMRT Link 5.1 software. Circular consensus sequences (CCSs) were generated from subread BAM files with the following parameters: min_length 50, max_drop_fraction 0.8, no_polish TRUE, min_zscore -9999.0, min_passes 2, min_predicted_accuracy 0.8, and max_length 15,000. CCS.BAM files were generated, which were then classified into full-length and non-full-length reads using pbclassify.py, ignorepolyA false, and minSeqLength 200. Non-full-length and full-length FASTA files were produced and fed into the cluster step, which performed isoform-level clustering, followed by final Arrow polishing, hq_quiver_min_accuracy 0.99, bin_by_primer false, bin_size_kb 1, qv_trim_5p 100, and qv_trim_3p 30. Additional nucleotide errors in consensus reads were corrected using the Illumina RNA sequencing data with the software LoRDEC (Salmela and Rivals, 2014). Any redundancy in corrected consensus reads was removed using the Cluster Database at High Identity with Tolerance (Fu et al., 2012) (link) (-c 0.95 -T 6 -G 0 -aL 0.00 -aS 0.99) to obtain final transcripts for subsequent analysis (Supplementary Figure S1).
3
Isoform Sequencing and Clustering
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The Iso-Seq library was prepared according to the Isoform Sequencing protocol (Iso-Seq) using the Clontech SMARTer PCR cDNA Synthesis Kit and the BluePippin Size Selection System protocol as described by Paci c Biosciences (PN 100-092-800-03). Sequence data were processed using the SMRTlink 5.0 software. The CCSs from subread BAM les (parameters: min length = 200, max drop fraction = 0.8, no polish TRUE, min z-score = -9999, min passes = 1, min predicted accuracy = 0.8, max length = 18,000) were classi ed into full length and non-full length reads using pbclassify.py script, ignore poly-A false, min Seq Length 200. Non-full length and full-length reads were then got into the clustering step, which does isoform-level clustering (ICE), followed by nal arrow polishing. Additional nucleotide errors in consensus reads were corrected using the Illumina RNAseq data with the software LoRDEC [70] .
4
Isoform Sequencing with Iso-Seq
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The Iso-Seq library was prepared according to the Isoform Sequencing protocol (Iso-Seq) using the Clontech SMARTer PCR cDNA Synthesis Kit and the BluePippin Size Selection System protocol as described by Paci c Biosciences (PN 100-092-800-03). Sequence data were processed using the SMRTlink 5.0 software. The CCSs from subread BAM les (parameters: min length = 200, max drop fraction = 0.8, no polish TRUE, min z-score = -9999, min passes = 1, min predicted accuracy = 0.8, max length = 18,000) were classi ed into full length and non-full length reads using pbclassify.py script, ignore poly-A false, min Seq Length 200. Non-full length and full-length reads were then got into the clustering step, which does isoform-level clustering (ICE), followed by nal arrow polishing. Additional nucleotide errors in consensus reads were corrected using the Illumina RNAseq data with the software LoRDEC [67].
5
Isoform Sequencing and Clustering
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The Iso-Seq library was prepared according to the Isoform Sequencing protocol (Iso-Seq) using the Clontech SMARTer PCR cDNA Synthesis Kit and the BluePippin Size Selection System protocol as described by Paci c Biosciences (PN 100-092-800-03). Sequence data were processed using the SMRTlink 5.0 software. The CCSs from subread BAM les (parameters: min length = 200, max drop fraction = 0.8, no polish TRUE, min z-score = -9999, min passes = 1, min predicted accuracy = 0.8, max length = 18,000) were classi ed into full length and non-full length reads using pbclassify.py script, ignore poly-A false, min Seq Length 200. Non-full length and full-length reads were then got into the clustering step, which does isoform-level clustering (ICE), followed by nal arrow polishing. Additional nucleotide errors in consensus reads were corrected using the Illumina RNAseq data with the software LoRDEC [70] .
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