Illumina HiSeq Sample Submission Form Project number (filled by FMSC) Project title (with one sentence) Project Description CONTACT INFORMATION Contact person(s) Name(s) Email(s) Address Phone Group leader/Principal investigator Name Email Address Phone Biocity Turku Research Programme (must be filled by Biocity Turku groups) INVOICING INFORMATION The laboratory part of the project will be invoiced after the raw data delivery. Internal invoice (University of Turku account) Name Department/Laboratory Cost pool (kustannuspaikka) TY project code: 26xxxxx External invoice (other than University of Turku account) Institution Department/Laboratory Address Cost pool (kustannuspaikka), if applicable VAT number PROJECT OPENING MEETING Date Participants (FMSC + customer) HiSeq Sample Submission Form 2015 Page 1 of 8 HiSeq PROTOCOL INFORMATION Read length Application type Number of lanes x 50 bp x 100 bp x 150 bp stranded mRNA-seq stranded total RNA-seq miRNA-seq smallRNA-seq ChIP-seq RRBS Bisulfite sequencing Targeted DNA-seq de novo genome sequencing genome resequencing No. of lanes used in sequencing: No. of samples per lane: Additional library preparation protocol information (e.g. special kit) SAMPLE INFORMATION Total number of samples (including biological replicates) Organism Sample type Tissue, origin Cells, origin Blood, origin Other, what Sample material DNA totalRNA poly(A) RNA smallRNA ready HiSeq library DNA / RNA extraction method Other possible pre-processing done to the samples (please specify all applied protocols) HiSeq Sample Submission Form 2015 Page 2 of 8 SAMPLE LIST Avoid spaces and any special characters in the sample names. Please use group names and sample names that will allow an easy interpretation of the name coding during data analysis. (Add rows if needed.) Also check the prices with FMSC if your total sample number has changed. Sample Sample name Sample group c, ng/l A260/A280 Volume, l Comments Index Pooling group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 GROUP DESCRIPTIONS Please give a short description of each sample group (add rows if needed). Please note that if you have changed your original sample setup from the one discussed with our bioinformaticians, you should contact them (bioinfo@btk.fi) to discuss the changes before continuing further. This concerns especially those projects where the data analysis service is used. Sample group Num. of biological replicates Description COMPARISON TO A SET OF PREVIOUS OR FUTURE RESULTS If you are planning to compare these results with a previous set of results produced using our service, please indicate here the time when the previous samples were processed If you are planning to compare these results directly with a possible future set of samples that will be processed using our service, please indicate this here and also give us some estimate when these samples will be processed. (It should be noted that after longer time the same equipment and protocols may not be available anymore, complicating the integration between earlier and later data set) HiSeq Sample Submission Form 2015 Page 3 of 8 DATA ANALYSIS SERVICE I would like to purchase the full bioinformatics data analysis service (please contact the bioinformatics team for pricing and details bioinfo@btk.fi) Data will be analyzed through collaboration with the FMSC bioinformatics group (as agreed previously). For service projects the bioinformatics cost will be invoiced after the results and the analysis report have been delivered. For collaboration projects at least 50% of the bioinformatics cost will be invoiced after the quality and the preliminary results have been delivered and the rest when the main results have been delivered or as agreed but latest within one year. I will analyze the data myself GENERAL OBJECTIVE OF DATA ANALYSIS FILL IN DIRECT COMPARISONS BETWEEN GROUPS if FMSC's bioinformatics team will analyze the data Use group names given in the sample information table. Remember that all groups do not necessarily need to be directly compared but the lists of differentially expressed genes between several independent comparisons (e.g. for different tissues) can also be compared. Comparison Comparison group 0 (example) KO (knock-out group) Baseline group WT (wild type group) 1 2 3 4 5 6 7 8 9 10 Paired samples Please indicate clearly (by e.g. sample naming) the samples that originate from the same biological individual as this information HiSeq Sample Submission Form 2015 Page 4 of 8 is needed in the analysis. Other information for the bioinformatic analysis (e.g. other factors linking certain samples together like handling day etc.) HiSeq Sample Submission Form 2015 Page 5 of 8 COLLABORATION WITH FMSC This page is to be filled by only those with whom collaboration has previously been agreed. Type of collaboration Lab Bioinformatics Financing of the project (What does each group contribute to material and personnel, given as detailed as possible, if relevant include payment schedule) FMSC members to be included as coauthors to publication(s) Other issues (e.g. Secrecy, place for storage of samples, time plan for discarding remains of samples, use of remaining samples for other purposes than this project.) HiSeq Sample Submission Form 2015 Page 6 of 8 Sample submission requirements and service work agreement The sample requirements and sample preparation instructions are described below for the different types of samples. FMSC will always check the quality of the samples (or libraries) prior to sequencing and the customer is contacted if there is a concern related to sample quality. Please note that samples may be processed even in the case they do not meet the quality requirements but then FMCS will not be able to guarantee the successful sequencing. Please do not hesitate to contact ngs@btk.fi for any further questions. Sending ready-made libraries for sequencing Ready-made libraries for HiSeq 2500 sequencing provided by user should have a concentration of at least 2 nM. The concentration of ready-made libraries should be 1-40 ng/µl in minimum of 10 µl volume. Library fragments should have compatible size distribution with selected read lenght in the range of 200700 bp and average fragment size should be 250-350 bp (for smallRNA libraries 120-170 bp) or according library preparation protocol’s recommendations. All library concentrations and fragment profiles should be similar in the same sample set. If several libraries are pooled in one lane, it is important to maintain color balance for each base of the index read being sequenced and thus select a set of compatible index sequences. Sending total RNA samples for library preparation and sequencing For RNAseq libraries total RNA samples should be diluted to 50-100 ng/µl, all in same concentration and minimum volume of 10 ul. At least 0.5 µg, preferably 1.0 µg, total RNA is required. For smallRNAseq libraries total RNA samples should be diluted to 200 ng/µl, all in same concentration and minimum volume of 10 µl. At least 1.5 µg, preferably 2.0 µg, total RNA is required. For good quality total RNA samples, the Agilent Bioanalyzer RIN value should be > 8.0 and all samples should have similar quality. Total RNA should be intact and pure. Using low quality or degraded RNA is not recommended with standard protocol as the use of degraded RNA can result in low yield, over-representation of the 3' ends of the RNA molecules, or failure of the protocol. RNA that has DNA contamination will result in underestimation of the amount of RNA used. To prevent this, DNase step is recommended, however, not required to be included with the RNA isolation method. If good quality material is not available, alternative workflows exist and can be discussed separately. When using low quality total RNA samples (RIN value < 7.0) for library preparation, all samples should be similar and equally degraded. Only fragments above 150 bp can be used for library preparation. FMSC does not guarantee results for samples with low RNA quality. Sending genomic DNA samples for library preparation and sequencing For DNAseq libraries genomic DNA samples should be diluted to 20-500 ng/ul, all in same concentration. Minimum of 0.5-3.0 µg genomic DNA is required, depending on the library preparation protocol. The ratio of absorbance at 260 nm to absorbance at 280 nm with values of 1.8–2.0 are considered indicative of pure DNA. Genomic DNA should be intact and pure, sample should be free of RNA or small nucleic acid fragments, such as nucleotides, or other contaminants. It is not recommend to use low quality or degraded DNA. DNA with RNA contamination, results in underestimation of the amount of DNA used. RNase step is recommended, however, not required to be included with the DNA isolation method. If there is not enough starting material or enough good quality material available, alternative workflows exist and can be discussed separately. However, FMSC does not guarantee results for samples with low DNA quality. HiSeq2500 sequencing run and result delivery Sample quality is controlled carefully during the library preparation and only good quality libraries will be sequenced. The typical yields are 120-180 x 106 paired-end or single-end reads per lane on HiSeq 2500, depending on the library type and quality. Sequencing will be rerun without extra costs in case of technical failure during the sequencing if it is not related to the nature or quality of the samples. The raw data in Illumina’s default format (fastq) will be downloadable within a week after the sequencing has been finished. Sample and data storage FMSC will discard the left over RNA/DNA sample material after 1 year. Please deliver only the amount of sample needed for the analysis. If extra material is for some reason sent and customer wishes to have it returned, it has to be agreed separately. Customer has to cover the possible costs related to the returning of the sample material. FMSC will store data only for a short time and the customer is responsible for storing the raw data after the project is finished and the data has been delivered to the customer. HiSeq Sample Submission Form 2015 Page 7 of 8 Send us this sample submission form both in print with your samples and electronically as an e-mail attachment to ngs@btk.fi. We require a signed copy of this document prior to processing the samples. Please check detailed sample submission instructions on our website at http://fmsc.btk.fi. As Principal Investigator on this project, I agree to: 1. Acknowledge the use of the Finnish Microarray and Sequencing Centre's analysis service in the materials and methods section in any resulting publications. 2. Send a reprint of each resulting publication to the Finnish Microarray and Sequencing Centre. 3. Include all involved FMSC members (see previous page) to my resulting publication(s) (where the analysis results are used) as coauthors if collaboration has been agreed with them. I will also send the manuscript for their review prior to submitting to a journal. _______________________ Date HiSeq Sample Submission Form 2015 _______________________ Signature Page 8 of 8