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Tender for Supply Of Next-Generation Sequencing (Ngs) Services For Whole-Genome Sequencing (Wgs) Of Plant S Les, Including Dna Extraction, 80 Gb (Q30) Trimmed Data Per S Le, Bulked Segregant Analysis (Bsa-Seq), Variant Discovery, Comparative Analysis, Polymorphism Detection, Gene Annotation, Candidate Region /Gene Identification And Linked-Marker Discovery. And S Le Transport (Dry Ice). , Next-Generation Sequencing (Ngs) Services For Whole-Genome Sequencing (Wgs) Of Plant Samples, Including Dna Extraction, 80 Gb (Q30) Trimmed Data Per Sample, Bulked Segregant Analysis (Bsa-Seq), Variant Discovery, Comparative Analysis, Polymorphism Detection, Gene Annotation, Candidate Region /Gene Identification And Linked-Marker Discovery. And Sample Transport (Dry Ice). Detailed Specification Service Required: Reference-Based Whole Genome Sequencing (Wgs) Including Dna Extraction, High-Quality Sequencing, Variant Discovery, Gene Annotation, And Comparative Genomic Analysis. Sample Type: Young Leaf Samples (Fresh Plant Tissue). Sample Transport: Transport From Field To Servicing Laboratory In Dry Ice, Until Extraction Of High-Quality Dna Suitable For Wgs. Cost Must Be Included. Sequencing Platform: Illumina Novaseq 6000 Or Latest Equivalent High-Throughput Platform. Date: Minimum =80 Gb High-Quality Trimmed Data (Q30), Where 20Gb Must Be From The Pacbio Platform Per Sample. Service And Analysis Requirement: 1. Dna Extraction, Qc And Bulk Preparation • High-Quality Genomic Dna Extraction From Each Of The 60 Individual Leaf Samples And 2-4 Parents. Separate Qc Record For Each Of The 60 Dna Samples. Normalisation Of Dna Concentration Followed By Equal-Quantity/Equimolar Pooling Of 15 Dnas Each Of Four Bulks. Documentation Of The Exact Contribution Of Each Individual Plant To Bulks 2. Wgs Data Generation And Quality Control •Trimmed And Quality-Filtered Paired-End Whole-Genome Sequencing Of 2-4 Bulks. Minimum =80 Gb Of High-Quality, Q30-Trimmed Data Per Bulk, Unless The Service Provider Proposes A Scientifically Justified Equivalent Coverage. Adapter Removal And Quality Filtering/Trimming. • Fastqc/Multiqc Or Equivalent Quality-Control Assessment. Raw-Read And Clean-Read Statistics, Q20/Q30, Gc Content, Read Count And Duplication Statistics. 3. Reference-Based Genome Analysis • Alignment Of Clean Reads To The Latest Appropriate Reference Genome. Reference Genome Accession/Version Must Be Reported. Reference-Based Mapping/Alignment Statistics. Total Reads Mapped And Percentage Mapped. Mapping Quality Statistics. Mean/Median Sequencing Depth. • Genome-Wide Coverage And Percentage Of Genome Covered At Relevant Depth Thresholds. Coverage/Depth Plots For Both Bulk-1 And Bulk-2. Reference-Based Assembly And Mapping Statistics: Total Reads Mapped, Depth And Genome Coverage, Percentage Of Genome Covered, Mapping Quality Metrics. 4. Variant Discovery / Polymorphism Detection • High-Confidence Snp And Indel Calling, Say, Between Bulk-1 And Bulk-2. • Genomic Coordinates Of All Detected Variants. Reference And Alternative Alleles For Both The Parental Genotypes And Discarding Of Common Alternate Alleles Between The Two Parental Genotypes. • Read Depth, Genotype/Allele Information And Quality Score For Each Variant.A Variant Was Retained Only When The Sequencing Depth Of Both Parents Was At Least 20 Reads. • At Least 90% Of Parent-1 Reads Were Required To Support The Reference Allele And Similarly, At Least 90% Of Parent-2 Reads Were Required To Support The Alternative Allele. •A Stringent 90% Allele-Support Threshold Must Be Applied At Each Informative Marker. The 90% Threshold Must Be Applied Only To The Parental Samples, Not To The Bulks. 5. Bulked Segregant Analysis (Bsa-Seq) Consider Both Snp And Indel Markers When The Parent-1-Derived Allele Was The Reference Allele • Snp-Index (Bulk1) = Parent-1 Derived Allele Reads In Bulk-1 / Total Bulk-1 Reads; Snp-Index(Bulk-2) = Parent-1-Derived Allele Reads In Bulk 2/ Total Bulk-2 Reads.; When The Parent-2-Derived Allele Was The Alternative Allele; Snp-Index (Bulk-1) = Parent-1 Derived Allele Reads In Bulk-1 / Total Bulk-1 Read; Snp-Index(Bulk-2) = Parent-1-Derived Allele Reads In Bulk 2/ Total Bulk-2 Reads.; Thus, The Calculated Index Always Represented The Proportion Of Reads Carrying The Allele Inherited From Parent-1, Rather Than Simply Representing The Frequency Of The Vcf Ref Or Alt Allele.; • ?snp-Index (Snp-Index Bulk 1 - Snp-Index Bulk-2) Calculation Across The Genome. A Positive ?snp-Index Therefore Indicated That The Parent-1-Derived Allele Was More Frequent In The Bulk-1 Than In The Bulk-2. Conversely, A Negative ?snp-Index Indicated That The Parent-1-Derived Allele Was More Frequent In The Bulk-2. Identification Of Statistically/Significantly Differentiated Genomic Regions/Peaks. Sliding Window: A Window Size Of 0.1 Mb (1,000,00 Bp) And A Step Size Of 10 Kb (100,00 Bp) Must Be Used.; Window Level-Qtl-Seq Stat: For Each Sliding Window, Mean Bulk-1 Parent-1-Derived Allele Index = Sum Of Bulk-1 Parent-1-Derived Allele Indices / Number Of Informative Variants; Mean Bulk-2 Parent-1-Derived Allele Index = Sum Of Bulk-2 Parent-1-Derived Allele Indices / Number Of Informative Variants. Mean ?snp-Index = Sum Of Individual ?snp-Index Values / Number Of Informative Variants. The Resulting Window-Level Dataset Therefore Summarized The Genomic Distribution Of Allele-Frequency Differences While Reducing The Influence Of Individual Marker-Level Fluctuations. 6. Variant Annotation And Functional Impact• Annotation Of Snps And Indels Against The Reference Gene Annotation.• Classification Into Intergenic, Upstream/Downstream, Intronic, Exonic And Other Relevant Categories.• Synonymous, Missense, Nonsense/Stop-Gain, Splice-Site And Other Predicted Effects Where Applicable.• Functional Impact Prediction/Prioritization Of Variants.• Identification Of Variants Located Within Or Near Candidate Genes. 7. Additional Deliverables • Summary Reports With All Statistical Outputs • Raw Data And Analysis Supporting Files Should Be Provided. • Fasta, Gff3, Vcf, Jpeg And Bam Files For All The Samples As Applicable • Detailed Compiled Report With Interpretations As Per The Instructions Of The Pi.
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