The file's text, extracted by GovTribe without its formatting.
SCOPE OF WORK for RNA sequencing for Dr. Imad Eujayl August 2019 Pg 3/3
Scope of Work:
Sequencing, Resequencing and Gene Expression analysis of Selected Sugar beet germplasm. We have 4 samples for whole genome sequencing and 24 samples for Gene Expression.
The genome size is 758MB.
CLR library.
Latest version of software.
Our timeline to delivery is 10 weeks.
Objectives of the Sequencing Project:
1. To improve the whole genome sequence (WGS) assembly of BwSeq-1 (the first USDA-ARS genome assembly-KDH13). This is targeting Beet curly top resistance genes
2. To establish the first whole genome assembly for KDH709-2 targeting resistance to rhizoctonia root rot disease.
3. To develop gene expression profiles for beet curly top and rhizoctonia root rot
Sequencing and resequencing KDH13,KDH709-2,KDH19-17, F1-13/19-17 are achieved through the following steps:
1. Samples and processing DNA extraction and perform quality control
2. Preparation of libraries (DNA fragments bank) for each sequencing analysis
3. Sequencing using different sequencing systems:
a. lllumina Sequencing platform: This is a sequencing system that produce millions of short (2x150 bp) DNA sequences (fragments).
b. PacBio sequencing platform: This is a sequencing system that produce long (8-15 KB) DNA sequences
4. Bioinformatics analysis:
a. Providing a FASTA formatted sequences
b. Develop a whole genome assembly
c. Submitting the assembly (BAMfiles) to the GenBank (www.ncbi.nlm.nih.gov)
Gene Expression Profiling:
1. Sample preparation and RNA processing:
2. Preparation of Libraries and quality control
3. RNA - sequencing using Illumina system
4. Bioinformatics: developing gene expression profiles based on pair-wise comparisons.
5. Submit RNASeq to the GenBank (www.ncbi.nlm.nih.gov).
References:
lllumina DNA sequencing: https:/ / www.illumina.co m/ systems/ se guencing-platforms.html PacBio DNA sequencing: https:/ / www.pacb.com/
| 1. | SERVICE 1 |
| Service |
| Quantity |
| DNA extraction, high molecular weight, PacBio |
| 1 |
| Library preparation, PacBio, 20 kb |
| 1 |
| PacBio Sequel sequencing |
| 3 |
| De novo assembly, PacBio, 401-999 Mb genome |
| 1 |
| Service Type |
| PacBlo Whole Genome Sequencing |
| Sample Type |
| Fresh frozen tissue |
| Sample Number |
| 1 sample(s) |
| Estimated Date Output |
| "Sequencing on 3 PacBio Sequel SMRT cell(s)" |
| Deliverables |
| Data summary report, BAM raw data files and de novo genome assembly |
| Level of Service |
| Research Use Only |
| Special Comments |
| Customer would like to complement WGS data from lllumina using |
PacBio 30x for breeding line KDH13
| 2. | SERVICE 2 |
| Service |
| Quantity |
| DNA extraction, high molecular weight, PacBio |
| 1 |
| Library preparation, PacBio, 20 kb |
| 1 |
| PacBio Sequel sequencing |
| 5 |
| De novo assembly, PacBio, 401-999 Mb genome |
| 1 |
| Service Type |
| PacBio Whole Genome Sequencing |
| Sample Type |
| . Fresh frozen tissue |
| Sample Number |
| 1 sample(s) |
| Estimated Data Output |
| "Sequencing on 5 PacBio Sequel SMRT cell(s)" |
| Deliverables |
| Data summary report, BAM raw data files, and de novo assembly |
| Level of Service |
| Research Use Only |
| Special Comments |
| WGS for KDH709-2 |
| 3. | SERVICE 3 |
| Service |
| Quantity |
| Library preparation, Illumina, RNA with rRna depletion |
| 24 |
| Illumina HiSeq 2x150b bp sequencing, single index |
| 2 |
| Standard RNA-Seq analysis |
| 24 |
| Data delivery, hard drive, Illumina |
| 1 |
| Service Type |
| Standard RNA-Seq |
| Sample Type |
| Total RNA (not derived from blood) |
| Sample Number |
| 24 sample(s) |
| Sequencing Configuration |
| Illumina HiSeq, 2x150 bp configuration, single index per lane |
| Estimated Data Output |
| ~350M raw paired-end reads per lane. |
| Quality Guarantee |
| ≥ 80% bases ≥ Q30 |
| Deliverables |
| Quality report, FASTQ files, data QC, trimming, mapping, differential gene expression, alternative splicing, and gene ontology analysis |
| Level of Service |
| Research Use Only |
| Special Comments |
| Map samples 1-18 to KDH13 (Reference genome generated in service 1, above). Map samples 19-24 to KDH709-2 (Reference genome generated in service 2, above) |
| 4. | SERVICE 4 |
| Service |
| Quantity |
| Library preparation, lllumina, DNA, value package |
| 2 |
| lllumina HiSeq 2x150 bp sequencing. single index, value package |
| 1 |
| Standard DNA-Seq analysis, value package |
| 2 |
| ServiceType |
| Whole genome sequencing |
| Sample Type |
| Frozen tissue |
| Sample Number |
| 2 sample(s} |
| Sequencing Configuration |
| lllumina HiSeq, 2x150bp configuration. single index, per lane |
| Estimated Data Output |
| -350M raw paired-end reads per lane |
| Quality Guarantee |
| ≥80% of bases ≥Q30 |
| Deliverables |
| Quality report. FASTQ files, data QC. trimming, mapping. SNP/INDEL, and intrachromosomal SV discovery |
| Level of Service |
| Research Use Only |
| Special Comments |
| Use reference genome from service 1, above, |
KDH13