Standard Products Nucleic Acid Sample Submission Guideline

Transcription

1 Standard Products Nucleic Acid Sample Submission Guideline Document NO.: Version NO.: SOP-SMM-028 A1 Effective Date:

2 Document NO.:SOP-SMM-028 Version NO.:A1 Page 1 of 19 CONTETS ABOUT THIS GUIDELINE TERMINOLOGY UNITS OF MEASUREMENT SAMPLE QUALITY INSPECTION METHOD RNA PRODUCTS TRANSCRIPTOME SEQUENCING Eukaryotic Transcriptome (BGISEQ-500) Eukaryotic transcriptome (Illumina Platform) Prokaryotic Transcriptome (Illumina Platform) Strand Specific Transcriptome (Illumina Platform) Transcriptome (PacBio) RNA-SEQ (QUANTIFICATION) RNA-Seq (Quantification) (BGISEQ-500) NON-CODING RNA LncRNA Small RNA (BGISEQ-500) OTHERS Immunization library CirRNA-Seq library DNA PRODUCTS WHOLE GENOME RESEQUENCING Whole Genome Resequencing (Illumina Platform) Human Whole Genome Resequencing (BGISEQ-500) Reduced-Representation Genome Sequencing, RRGS Exon sequencing (Illumina Platform) Exon sequencing (BGISEQ-500) Target region sequencing (Illumina Platform) DE NOVO SEQUENCING De novo sequencing (Illumina Platform) De novo sequencing (PacBio) METHYLATION SEQUENCING Whole Genome Bisulfite Sequencing ChIP-Seq(BGISEQ-500) Target Region Bisulfite Sequencing GENOTYPING... 15

3 Document NO.:SOP-SMM-028 Version NO.:A1 Page 2 of Genotyping (Affymetrix) META PRODUCTS Metagenomic Survey Meta rdna Amplicon Sequencing OTHERS Immunization library SELF-PREPARED LIBRARY TRANSCRIPTOME/ QUANTIFICATION PREMADE LIBRARY SMALL RNA PREMADE LIBRARY CHIP PREMADE LIBRARY EXOME CAPTURE PREMADE LIBRARY DNA SHORT FRAGMENT(0-499BP) PREMADE LIBRARY DNA LONG FRAGMENT ( BP) PREMADE LIBRARY DNA LARGE FRAGMENT (5 800BP) PREMADE LIBRARY METHYLATION PREMADE LIBRARY HISEQ SEQUENCING PRIMERS... 19

4 Document NO.:SOP-SMM-028 Version NO.:A1 Page 3 of 19 About This Guideline This guideline details sample quality standards for RNA products, DNA products, and selfprepared libraries for BGI Scientific Research Services. Declaration For biological agents that require handling in biosafety level 3 or level 4 facilities, purified nucleic acid samples from those biological agents are acceptable. However, any types of BSL3 or BSL4 tissue samples will not be accepted. For biological agents that require handling in biosafety level 1 or level 2 facilities, clients may send tissue samples to BGI only after discussing with our representatives about pathogenicity and infectiousness of the samples. For any pathogenic or infectious samples, or potential pathogenic or infectious samples (e.g. tissue, blood, bacterium etc.), they must be shipped in packaging and clearly labeled on the sample tubes or bags following instruct below: Primary receptacles must be leak-proof. Secondary packaging must be leak-proof and capable of protecting the primary receptacles during transport condit. Enclose a detailed itemized list of contents along with a note stating Pathogenic substance, please handle with caution between the secondary packaging and the outer packaging. Outer packaging must be rigid and properly labeled. BGI representatives must notify the sample management center with detailed information (package tracking number, number of sample, sample type, handling precaution, contact of client, and estimated arrival time) once the samples are sent. Should you have any enquiries, please contact our customer service at info@genomics.cn or

5 Document NO.:SOP-SMM-028 Version NO.:A1 Page 4 of 19 1 Terminology This document uses the following terms: 1.1 Units of measurement 1) v:volume, sample volume. 2) m:total Mass, total amount of DNA/RNA 3) c:concentration, concentration of DNA/RNA 4) M:Molar Concentration, molar concentration of DNA / RNA (exact quantification) 5) size:fragment size, fragment size of DNA / RNA 6) RIN:RNA Integrity Number, RIN is an algorithm for assigning integrity values to RNA measurements 7) 28S/18S:The ratio of 28S to 18S, which reflects the integrity of Eukaryotes RNA 8) 23S/16S:The ratio of 23S to 16S, which reflects the integrity of Prokaryotes RNA 9) OD260/280:The ratio of OD260 to OD280, which reflects the purity of DNA/RNA 10) OD260/230:The ratio of OD260 to OD230, which reflects the purity of DNA/RNA 1.2 Sample quality inspection method 1) Invitrogen Qubit Fluorometer 2) Thermo Fisher NanoDrop TM ND-1000/ND ) Agilent 2100 Bioanalyzer, 4) Caliper LabChip GX 5) Agarose Gel Electrophoresis.

6 Document NO.:SOP-SMM-028 Version NO.:A1 Page 5 of 19 2 RNA Products Customers need to provide sample analysis in one or more forms of NanoDrop TM, AGE, or Agilent 2100, etc. Please carefully purify the sample to avoid residue of polysaccharide, protein and exonuclease; the sample must indicate the solvent composition. Please note that BGI does not receive any infectious RNA samples. Customers need to send cdna samples via reverse transcription of RNA. For more details, please contact our BGI representative. The quality of the sample is based on the BGI quality test. Customers need to understand that the test results may differ from theirs due to the difference in locat, equipment and operators in the tests. Customers must send sample of at least 200ng more than that stated in each products standard sample amount requirement due to certain amount of sample will be consumed during the quality control process. It is strongly recommended to prepare samples at least two times the standard requirement, otherwise samples may be evaluated as unqualified in quality control process and therefore delay the progress of the project. BGI only receives 1.5mL/2.0mL EP tubes, containing sample volume of μL per tube (30μL is recommended). According to the experimental requirements, the original sample may be diluted if the sample volume is less than 15μL. 2.1 Transcriptome sequencing Eukaryotic Transcriptome (BGISEQ-500) Transcriptome Sample type Mass Concentration RIN (Plant / Fungi) 28S/18S (23S/16S) or DV 200 1μg 40ng/μL RIN S/18S 1.0 Baseline and 5S Sample Purity (Animal) (Human / Rat) 1μg 40ng/μL RIN S/18S ng 20ng/μL RIN S/18S 1.0 Baseline is smooth; 5S is normal (Insect) 1μg 40ng/μL N/A Eukaryotic transcriptome (Illumina Platform)

7 Document NO.:SOP-SMM-028 Version NO.:A1 Page 6 of 19 Eukaryotic Transcriptome Sample type Mass Concentration RIN (Plant / Fungi) (Animal) (Human / Rat) (Insect) 28S/18S or DV 200 1μg 40ng/μL RIN S/18S 1.0 1μg 40ng/μL RIN S/18S ng 20ng/μL RIN S/18S 1.0 1μg 40ng/μL N/A FFPE RNA 200ng 70ng/μL RIN 2.0 DV % mrna purified with oligo(dt) or RNA depleted of rrna 30ng 6ng/μL dscdna 1μg 15ng/μL Fragment size is greater than 1Kb. Baseline and 5S Baseline is smooth; 5S is normal Sample Purity Prokaryotic Transcriptome (Illumina Platform) Prokaryotic Transcriptome Sample type Mass Concentration RIN 23S/16S Baseline and 5S Sample Purity 1μg 40ng/μL RIN S/16S Strand Specific Transcriptome (Illumina Platform) Baseline is smooth; 5S is normal Strand Specific Transcriptome Sample type Mass Concentration RIN 28S/18S(23S/16S) Baseline and 5S Sample Purity (Plant / Fungi) 1μg 40ng/μL RIN S/18S 1.0 Baseline is smooth; 1μg 40ng/μL RIN S/18S 1.0 (Animal) 5S is normal (Human / Rat) 200ng 20ng/μL RIN S/18S 1.0 Not contaminated

8 Document NO.:SOP-SMM-028 Version NO.:A1 Page 7 of 19 (Prokaryotic) (Insect) mrna purified with oligo(dt) or RNA depleted of rrna 1μg 40ng/μL RIN S/16S 1.0 1μg 40ng/μL N/A 30ng 6ng/μL Fragment size is greater than 1Kb; rrna<10%. Not contaminated Transcriptome (PacBio RSII&Sequel) Transcriptome Sample type Mass Concentration RIN 28S/18S Baseline and 5S Sample Purity 1μg 285ng/μL RIN S/18S 1.4 Baseline is smooth; 5S is normal OD260/ ; OD260/ RNA-Seq (Quantification) RNA-Seq (Quantification) (BGISEQ-500) RNA-Seq (Quantification) Sample type Mass Concentration RIN 28S/18S Baseline and 5S Sample Purity (Plant / Fungi) (Animal) (Human / Rat) (Insect) mrna purified with oligo(dt) or RNA depleted of rrna 1μg 40ng/μL RIN S/18S 1.0 1μg 40ng/μL RIN S/18S ng 20ng/μL RIN S/18S 1.0 1μg 40ng/μL N/A Baseline is smooth; 5S is normal 100ng 15ng/μL Fragment size is greater than 1Kb; rrna<10%.

9 Document NO.:SOP-SMM-028 Version NO.:A1 Page 8 of Non-coding RNA LncRNA LncRNA Sample type Mass Concentration RIN (Plant / Fungi) (Animal) (Human / Rat) (Insect) 28S/18S or DV 200 1μg 40ng/μL RIN S/18S 1.0 1μg 40ng/μL RIN S/18S ng 20ng/μL RIN S/18S 1.0 1μg 40ng/μL N/A FFPE RNA 1μg 70ng/μL RIN 2.0 DV % N/A mrna purified with oligo(dt) or RNA depleted of rrna Baseline and 5S Baseline is smooth; 5S is normal 30ng 6ng/μL Fragment size is greater than 1Kb; rrna<10%. Sample Purity Small RNA (BGISEQ-500) Small RNA Sample type Mass Concentration RIN (Plant / Fungi) (Animals) (Prokaryotes) (Insect) 28S/18S (23S/16S) or DV 200 1μg 50ng/μL RIN S/18S 1.3 1μg 50ng/μL RIN S/18S 1.5 1μg 50ng/μL RIN S/16S 1.5 1μg 50ng/μL N/A FFPE RNA 1μg 50ng/μL RIN 2.0 DV % N/A Baseline and 5S Baseline is smooth; 5S is normal Sample Purity

10 Document NO.:SOP-SMM-028 Version NO.:A1 Page 9 of 19 Small RNA (<200nt) Small RNA of Plasma/serum 100ng 5ng/μL N/A N/A 20ng 2ng/μL N/A N/A 2.4 Others Immunization library Immunization library is mainly for the T cell β chain CDR3 region analysis. For other chains submission requirements, please contact the BGI representative in charge of the project for further information. Note: For tissue samples, if the lymphocyte content is low, it cannot be amplified. Immunization library Sample type Mass Concentration RIN 28S/18S Baseline and 5S Sample Purity Sorted T cells RNA / PBMCs RNA Whole blood RNA/ Tissue RNA 500ng 35ng/μL RIN S/18S 1.0 1μg 70ng/μL RIN S/18S 1.0 Baseline is smooth; 5S is normal CirRNA-Seq library CirRNA-Seq library Sample type Mass Concentration RIN 28S/18S Baseline and 5S Sample Purity (Plant) (Animal) 5μg 40ng/μL RIN S/18S 1.0 5μg 40ng/μL RIN S/18S 1.0 Baseline is smooth; 5S is normal 3 DNA Products Customers need to provide sample analysis in one or several of the following methods: Qubit, AGE, or Agilent 2100, etc. Please carefully purify the sample to avoid residue of polysaccharide, protein and exonuclease. The solvent composition of the sample must be indicated.

11 Document NO.:SOP-SMM-028 Version NO.:A1 Page 10 of 19 The quality of the sample is based on the BGI quality test. Customers need to understand that the test results may differ from theirs due to the difference in locat, equipment and operators in the tests. Customers must send sample of at least 50ng more than that stated in each products standard sample amount requirement due to certain amount of sample will be consumed during the quality control process. It is strongly recommended to prepare samples at least two times the standard requirement, otherwise samples may be evaluated as unqualified in quality control process and therefore delay the progress of the project. BGI only receives 1.5mL/2.0mL EP tubes, containing sample volume of μL per tube (30μL is recommended). According to the experimental requirements, the original sample may be diluted if the sample volume is less than 15μL. 3.1 Whole Genome Resequencing Whole Genome Resequencing (Illumina Platform) Whole Genome Resequencing Genomic DNA Plasmid, PCR product, etc. Normal library 1μg 12.5ng/μL PCR free library 10μg 30ng/μL Normal library 1μg 12.5ng/μL PCR free library (no fragmentation 3μg is needed) 30ng/μL The band shown on gel electrophoresis has little degradation, or of fragment size greater than 20kb. The band shown on gel electrophoresis is clear without smear. No contamination with RNA, ; colorless and transparent; non-sticky Human Whole Genome Resequencing (BGISEQ-500) Human Whole Genome Resequencing Genomic DNA 1μg 12.5ng/μL Reduced-Representation Genome Sequencing, RRGS The band shown on gel electrophoresis RNA, has little degradation, or of fragment size ; colorless and greater than 20kb. transparent; non-sticky.

12 Document NO.:SOP-SMM-028 Version NO.:A1 Page 11 of RAD-Seq RAD Library Genomic DNA 1μg 20ng/μL Genotyping by Sequencing The band shown on gel electrophoresis RNA, has little degradation, or of fragment size ; colorless and greater than 20kb. transparent; nonsticky. GBS Library Genomic DNA 300ng 10ng/μL Exon sequencing (Illumina Platform) The band shown on gel electrophoresis RNA, has little degradation, or of fragment size ; colorless and greater than 20kb. transparent; non-sticky. Low input library construction (<1μg) is an option in exon sequencing, but customers need to consider carefully as there is certain risk of failure in library construction. For low input library construction, sample (not FFPE sample) need to be of mass greater than 200ng, concentration greater than 2.5 ng/μl, and the band shown on gel electrophoresis has little degradation. If Agilent SureSelect QXT kit for low input library construction is used, sample need to be of mass greater than 50ng along with concentration greater than 25 ng/μl. For low input library construction, FFPE DNA samples have a lower successful rate than non- FFPE DNA samples. FFPE DNA samples need to be of mass greater than 200ng, concentration greater than 2.5 ng/μl, and fragment size greater than 500bp. Exon sequencing Genomic DNA 1μg 12.5ng/μL The band shown on gel electrophoresis has little degradation, or of fragment size greater than 20kb. FFPE DNA 1μg 12.5ng/μL Fragment size greater than 500bp. RNA, ; colorless and transparent; non-sticky.

13 Document NO.:SOP-SMM-028 Version NO.:A1 Page 12 of Exon sequencing (BGISEQ-500) Exon sequencing Genomic DNA 1μg 12.5ng/μL Target region sequencing (Illumina Platform) The band shown on gel electrophoresis has little degradation, or of fragment size greater than 20kb. RNA, ; colorless and transparent; non-sticky. Low input library construction (<1μg) is an option in target region sequencing, but customers need to consider carefully as there is certain risk of failure in library construction. For low input library construction, sample (not FFPE sample) need to be of mass greater than 200ng, concentration greater than 2.5 ng/μl, and the band shown on gel electrophoresis has little degradation. If Agilent SureSelect QXT kit for low input library construction is used, sample need to be of mass greater than 50ng along with concentration greater than 25 ng/μl. For low input library construction, FFPE DNA samples have a lower successful rate than non- FFPE DNA samples. FFPE DNA samples need to be of mass greater than 200ng, concentration greater than 2.5 ng/μl, and fragment size greater than 500bp. Exon sequencing Genomic DNA 1μg 12.5ng/μL The band shown on gel electrophoresis has little degradation, or of fragment size greater than 20kb. FFPE DNA 1μg 12.5ng/μL Fragment size is greater than 500bp RNA, ; colorless and transparent; non-sticky. 3.2 De novo sequencing Quantification tests for DNA samples with larger fragment need to be based on Qubit or AGE while quantification by NanoDrop TM is not recommended De novo sequencing (Illumina Platform)

14 Document NO.:SOP-SMM-028 Version NO.:A1 Page 13 of 19 De novo sequencing Genomic DNA 800bp library 1μg 12.5ng/μL The band shown on gel electrophoresis has little 800bp PCR free library 10μg 30ng/μL 2-6Kb mate pair library 20μg 100ng/μL degradation, or of fragment size greater than 20kb. 10Kb mate pair library 30μg 66.6ng/μL The band shown on gel electrophoresis has little 20Kb mate pair library 40μg 88.8ng/μL 40Kb mate pair library 50μg 110ng/μL degradation, or of fragment size greater than 40kb. No contamination with RNA, ; colorless and transparent; non-sticky Plasmid, PCR 800bp library 800bp PCR free library 1μg 12.5ng/μL product, etc. (no fragmentation is 3μg 30ng/μL needed) The band shown on gel electrophoresis is clear without smear De novo sequencing (PacBio) Quality requirements for PacBio sequencing is very high. Please ensure the quality of customers samples meet the following requirements. Customers should also purify their sample carefully in order to remove polysaccharide, protein and exonuclease residues. Detailed solvent components should be provided. DNA samples should be dissolved in buffer without EDTA, chelating agent, divalent metal cat, denaturants, detergents, or visual fluorescent dyes. DNA samples should not be recovered from EB stained gel. Samples cannot be frozen and thawed repeatedly, stored at high temperature or in extreme ph condition (ph<6 or ph>9). They should be preserved at -80 and shipped to BGI with dry ice. For degraded genomic samples, it is recommended to re-extract the DNA from tissue. If the sample is scarce, it can be treated accordingly depending on the degradation level; however, quality of library construction or sequencing data may be poor. Library construction can still be performed if the fragment size of sample is greater than 10Kb. However, customers need to understand the risk of failure in library construction or poor quality in sequencing. Degraded sample can undergo size selection for larger fragment by magnetic beads or blue pippin. The quantity needed will be adjusted according to the degradation level. However, not all of the small fragment can be eliminated due to

15 Document NO.:SOP-SMM-028 Version NO.:A1 Page 14 of 19 different sample quantity or quality. Customers need to understand the risk of failure in library construction or poor quality in sequencing. For samples have abnormal 260/280 ratio or 260/230 ratio, or Qubit-HS/Nanodrop<0.5, they can be purified using magnetic beads of column followed by a second quality check PacBio Sequel De novo sequencing (PacBio Sequel) Sample type Mass Concentration OD Integrity (AGE) Sample Purity Genomic DNA Plant / Animal Microorganism 20Kb 2.5μg/Cell ( 13μg/Sample) 60ng/μL 13μg / Sample 60ng/μL The band shown on OD260/280: gel electrophoresis has little OD260/230: degradation, or of fragment size greater than 40kb. No contamination with RNA, ; colorless and transparent; non-sticky PacBio RSII De novo sequencing (PacBio RSII) Sample type Mass Concentration OD Integrity (AGE) Sample Purity Genomic DNA Plant / Animal 20Kb 8μg 60ng/μL Microorganism 8μg 60ng/μL OD260/280: OD260/230: OD260/280: OD260/230: The band shown on gel electrophoresis has little degradation, or of fragment size greater than 40kb. No contamination with RNA, protein or salt ; colorless and transparent; nonsticky 3.3 Methylation Sequencing Whole Genome Bisulfite Sequencing Whole Genome Bisulfite Sequencing

16 Document NO.:SOP-SMM-028 Version NO.:A1 Page 15 of 19 Genomic DNA 1μg 12.5ng/μL ChIP-Seq (BGISEQ-500) The band shown on gel electrophoresis RNA, ; has little degradation, or of fragment size colorless and transparent; greater than 20kb. non-sticky. ChIP-Seq Sample type Mass Concentration Integrity (Agilent 2100) Sample Purity Genomic DNA 10ng 1ng/μL Fragment size is between bp. RNA, ; colorless and transparent; non-sticky Target Region Bisulfite Sequencing Target Region Bisulfite Sequencing Human Genomic DNA TBS Library (Agilent) TBS Library (NimbleGen EZ) 3μg 37.5ng/μL The band shown on gel electrophoresis has little degradation, or of 1μg 12.5ng/μL fragment size greater than 20kb. No contamination with RNA, protein or salt ; colorless and transparent; nonsticky. 3.4 Genotyping Genotyping (Affymetrix) Genotyping Genomic DNA 500ng 50ng/μL The band shown on gel electrophoresis RNA, ; has little degradation, or of fragment size colorless and transparent; greater than 20kb. non-sticky. 3.5 Meta products Metagenomic Survey

17 Document NO.:SOP-SMM-028 Version NO.:A1 Page 16 of 19 Metagenomic Survey Genomic DNA 1μg 12.5ng/μL Meta rdna Amplicon Sequencing The band shown on gel electrophoresis has little degradation RNA, ; colorless and transparent; non-sticky. Meta rdna Amplicon Sequencing includes Meta rdna V3, V6, V4, V1-V3, V3-V4, V4-V5, V5-V6,ITS1, ITS2. reg amplicon library construction (18s and other regional or non-bgi standard method for building the library need to communicate with the customization team). The key step of library construction is PCR amplification, which is affected by many factors such as purity, salt, pigment, and humic acid. Therefore, the quality of Meta rdna Amplicon samples will be determined after library construction with PCR amplification. All genomic DNA samples will be proceeded for library construction. If the first attempt fails, an optimized library construction will be performed acquiescently when the remaining sample is more than 200ng. No more library construction will be performed with the remaining sample is less than 200ng. If the first library construction is not successful, the sample is classified as unqualified. Customers may require a second library construction with additional fee in this case, but the success rate will not be guaranteed. Meta rdna Amplicon Sequencing Meta rdna Amplicon Meta 16S rdna Amplicon PCR-free Library Genomic DNA PCR products 0ng (above 50ng recommended) 3μg 0ng/μL 30ng/μL Sample must be genomic DNA. The band shown on gel electrophoresis is clear without smear. RNA, ; colorless and transparent; non-sticky. 3.6 Others Immunization library

18 Document NO.:SOP-SMM-028 Version NO.:A1 Page 17 of 19 Immune Repertoire Sequencing is suitable for Human T cell β chain CDR3 region analysis. If sequencing for other chains is needed, please inquire with the BGI representative in charge of the project. Note: If the lymphocytes content in tissue sample is too low, PCR may fail. Immunization library Sorted T cells DNA / PBMCs DNA Whole blood DNA/ Tissue DNA 1.5μg 0.5μg 30ng/μL 85ng/μL The band shown on gel electrophoresis has little degradation, or of fragment size greater than 20kb. RNA, ; colorless and transparent; nonsticky. 4 Self-prepared Library Customers need to provide sample analysis in one or several of the following methods: Qubit, AGE, or Agilent 2100, and Caliper LabChip GX. Agilent DNA 1000 kit is recommended for Agilent2100, and HT DNA 1K Lab Chip is recommended for Caliper Lab Chip GX. 4.1 Transcriptome/ Quantification Premade Library Transcriptome/ Quantification Premade Library Sequencing platform Quantity Concentration Integrity HiSeq 2500 platform * 10-3 pmol 5.2nM than 600bp. Single peak should be observed. HiSeq 4000 platform 200 * 10-3 pmol 10nM than 500bp. Single peak should be observed. 4.2 Small RNA Premade Library Transcriptome/ Quantification Premade Library Sequencing platform Quantity Concentration Integrity HiSeq 2500 platform 70.5 * 10-3 pmol 3nM than 600bp. Single peak should be observed.

19 Document NO.:SOP-SMM-028 Version NO.:A1 Page 18 of 19 HiSeq 4000 platform 200 * 10-3 pmol 10nM than 500bp. Single peak should be observed. 4.3 ChIP Premade Library ChIP Premade Library Sequencing platform Quantity Concentration Integrity HiSeq 2500 platform 70.5 * 10-3 pmol 5nM HiSeq 4000 platform 200 * 10-3 pmol 10nM than 600bp. Single peak should be observed. than 500bp. Single peak should be observed. 4.4 Exome Capture Premade Library Exome Capture Premade Library Sequencing platform Quantity Concentration Integrity HiSeq 2500 platform * 10-3 pmol 5.8nM than 600bp. Single peak should be observed. HiSeq 4000 platform 200 * 10-3 pmol 10nM than 500bp. Single peak should be observed. 4.5 DNA Short Fragment (0-499bp) Premade Library DNA Short Fragment(0-499bp) Premade Library Sequencing platform Quantity Concentration Integrity HiSeq 2500 platform * 10-3 pmol 5.4nM than 600bp. Single peak should be observed. HiSeq 4000 platform 200 * 10-3 pmol 10nM than 500bp. Single peak should be observed. 4.6 DNA Long Fragment ( bp) Premade Library DNA Long Fragment ( bp) Premade Library Sequencing platform Quantity Concentration Integrity

20 Document NO.:SOP-SMM-028 Version NO.:A1 Page 19 of 19 HiSeq 2500 platform * 10-3 pmol 5.5nM HiSeq 4000 platform 200 * 10-3 pmol 10nM than 600bp. Single peak should be observed. than 500bp. Single peak should be observed. 4.7 DNA Large Fragment (5 800bp) Premade Library DNA Large Fragment (5 800bp) Premade Library Sequencing platform Quantity Concentration Integrity HiSeq 2500 platform * 10-3 pmol 4.5nM HiSeq 4000 platform 200 * 10-3 pmol 10nM than 600bp. Single peak should be observed. than 500bp. Single peak should be observed. 4.8 Methylation Premade Library Methylation Premade Library Sequencing platform Quantity Concentration Integrity HiSeq 2500 platform 94 * 10-3 pmol 4nM HiSeq 4000 platform 200 * 10-3 pmol 10nM than 600bp. Single peak should be observed. than 500bp. Single peak should be observed. 4.9 HiSeq Sequencing Primers HiSeq Sequencing Primers Primer name Volume Remark Read 1 primer Index primer Read 2 primer 20μL/lane+20μL backup 20μL/lane+20μL backup 20μL/lane+20μL backup For primer in solution, the concentration should be at least 100μM.