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Iso-Seq analysis

Demultiplexed hifi reads (Q20, single-molecule resolution) from lima [https://github.com/pacificbiosciences/barcoding/ https://github.com/pacificbiosciences/barcoding/] were further processed using Isoseq3 (v3.2.2) [https://github.com/PacificBiosciences/IsoSeq] refinement and clustering steps to generate the HQ (Full-length high quality i.e., predicted accuracy ≥Q20) transcripts [Table 1]. Next, we mapped these transcripts against GRCh38 (v33 p13) [2] using Minimap2 long read alignment tools [1] (v2.24-r1122; commands: minimap2 -t 8 -ax splice:hq -uf --secondary=no -C5 -O6,24 -B4 GRCh38.v33p13.primary_assembly.fa sample.polished.hq.fastq.gz). The table 2 shows the basic statistics of the alignment of each sample [HG002: NA27730, NA24385 and NA26105]. Next, we performed cDNA_cupcake v28.0.0 [https://github.com/Magdoll/cDNA_Cupcake] workflow to collapse the redundant isoforms from bam, followed by filtering the low counts isoforms by 10 and filter away 5' degraded isoforms as they are not biologically significant. Next, sqanti3 v5.0 [3] tool was used to generate the final corrected fasta transcripts and gff [Table 3] along with the isoform classification reports. The external databases including reference data set of transcription start sites (refTSS), list of polyA motif, tappAS-annotation and Genecode GRCh38 annotation were utilized during the isoform classification by Sqanti3. Finally, IsoAnnotLite (v2.7.3) [https://isoannot.tappas.org/isoannot-lite/] analysis was performed to annotate the gtf file from sqanti3.

Table 1. Statistics of HQ fastq reads. Samples num_seqs min_len avg_len max_len N50 Q20(%) Q30(%) NA26105 (HG002.3) 205,590 51 3298.8 14887 3817 99.95 99.92 NA27730 (HG002.2) 205,884 52 3093.6 12524 3695 99.96 99.93 NA24385/HG002 (HG002.1) 411,349 50 2171.2 10767 2675 99.96 99.93

Table 2. Statistics of isoforms mapped with Minimap2. Samples Total Reads AlnPerc (%) Mapped Reads Unmapped reads NA26105 MM2 99.04 203607 1983 NA27730 MM2 99.35 204553 1331 NA24385 MM2 99.65 409905 1444

Table 3. Basic Statistics of transcripts discovered by Sqanti3 filtered. Sample # of Gene # of transcript # of exon NA26105 5433 7632 72940 NA27730 5459 7765 74906 NA24385/HG002 5656 9710 51196

Reference:

  1. Heng Li, Minimap2: pairwise alignment for nucleotide sequences, Bioinformatics, Volume 34, Issue 18, September 2018, Pages 3094–3100, https://doi.org/10.1093/bioinformatics/bty191
  2. Adam Frankish and others, GENCODE 2021, Nucleic Acids Research, Volume 49, Issue D1, 8 January 2021, Pages D916–D923, https://doi.org/10.1093/nar/gkaa1087
  3. Pardo-Palacios FJ, Arzalluz-Luque A et al. SQANTI3: curation of long-read transcriptomes for accurate identification of known and novel isoforms. doi: 10.1101/2023.05.17.541248. PMID: 37398077; PMCID: PMC10312485. image