From 45807f2f825b29a57f0e8a19c5d354b976d2059b Mon Sep 17 00:00:00 2001
From: Rupesh Kesharwani <41694905+unique379r@users.noreply.github.com>
Date: Fri, 1 Sep 2023 11:49:00 -0500
Subject: [PATCH] Create workflow.md

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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](https://github.com/collaborativebioinformatics/isocomp/assets/41694905/60b9790a-3caf-499c-b799-bcd819ec0e6c)