The subject of the public procurement is various types of sequencing services based on reading the order of nucleotides in DNA molecule chains (both DNA-Seq and RNA-Seq), as well as other supplementary services related to the preparation and quality control of samples for these analyses and the analysis of acquired data. This public procurement is divided in accordance with § 35 ZZVZ into 8 parts.

LOT-0001
Part 1 – Paired sequencing of a single library/pool 2×150 bp with a quantity of at least 1.5 Gb and an average quality of nucleobase identification for each individual read (sequence) of at least 30 or higher (Phred quality score Q30) for at least 70% of reads.
The sequencing includes basic quality control, e.g., measured on a Bioanalyzer or similar device, sequencing, and basic demultiplexing of the obtained data, if this step is relevant, without further bioinformatics processing or data evaluation, according to the description of the individual parts in point 1.2 of the tender documentation.
Sequencing + supplementary services: The supplied genomic DNA sample will be checked, and its suitability for sequencing will be determined. The supplier will prepare a library from the sample. This library will then be sequenced with a read length of 150 bp in paired mode (2×150 bp) and with a minimum output of 1.5 Gb and quality defined in the description of Part 1. As sequencing output, two FASTQ files containing forward and reverse read will be delivered to the client – ideally via an online platform.
Expected volume over 24 months – 133 samples.

LOT-0002
Part 2 – Paired sequencing of a library/pool 2×150 bp with a quantity of at least 50 Gb and an average quality of nucleobase identification for each individual read (sequence) of at least 30 or higher (Phred quality score Q30) for at least 70% of reads.
The sequencing includes basic quality control, e.g., measured on a Bioanalyzer or similar device, sequencing, and basic demultiplexing of the obtained data, if this step is relevant, without further bioinformatics processing or data evaluation, according to the description of the individual parts in point 1.2 of the tender documentation.
Sequencing + supplementary services: This example consists of 15 individual RNA samples, isolated in the client's laboratory. After delivery to the supplier, their quality will be checked, and suitability for sequencing will be determined. The supplier will prepare a pool of libraries (sequencing pool) with unique indexes for each sample. This pool will then be sequenced with a read length of 150 bp in paired mode (2×150 bp) and with a minimum output of 50 Gb and quality defined in the description of Part 2. The obtained data will then be evaluated bioinformatically. This evaluation includes identification, quantification, and mapping of transcripts and comparison of gene expression in samples or groups of samples (as specified by the client) and determination of the extent (multiples of expression) and significance of these changes. These results will be provided to the client or made available for download from the supplier's server, at minimum in the form of raw sequencing data, a comprehensive table with results, and tables and graphs documenting differences in expression.
Expected volume over 24 months – 12 pools.

LOT-0003
Part 3 – Paired sequencing of a library/pool 2×150 bp with a quantity of at least 240 Gb and an average quality of nucleobase identification for each individual read (sequence) of at least 30 or higher (Phred quality score Q30) for at least 70% of reads.
The sequencing includes basic quality control, e.g., measured on a Bioanalyzer or similar device, sequencing, and basic demultiplexing of the obtained data, if this step is relevant, without further bioinformatics processing or data evaluation, according to the description of the individual parts in point 1.2 of the tender documentation.
Sequencing + supplementary services: This example includes sequencing a pool consisting of several libraries, prepared using the Chromium Next GEM Single Cell 3´ or 5´ Reagent kit, where DNA fragments are equipped with specific adapter sequences necessary for sequencing on the Illumina platform or similar. Quality control of the input material (1 pool) is required using capillary electrophoresis, e.g., Agilent or similar, and the actual sequencing with a read length of 150 bp in paired mode with a yield of at least 240 Gb and in quality defined in the description of Part 3. The sequencing output will then be demultiplexed and at the same time, sequencing adapters will be removed from the data (according to information from the client), and for each library, two FASTQ files containing forward and reverse read will be delivered to the client – ideally via an online platform.
Expected volume over 24 months – 9 pools.

LOT-0005
Part 4 – Paired sequencing of a library/pool 2×250 bp with a quantity of at least 5 Gb and an average quality of nucleobase identification for each individual read (sequence) of at least 30 or higher (Phred quality score Q30) for at least 60% of reads.
The sequencing includes basic quality control, e.g., measured on a Bioanalyzer or similar device, sequencing, and basic demultiplexing of the obtained data, if this step is relevant, without further bioinformatics processing or data evaluation, according to the description of the individual parts in point 1.2 of the tender documentation.
Sequencing + supplementary services: The supplied sequencing pool containing several (3-8) bacterial genomes (libraries defined, e.g., by TrueSeq adapters) will be checked (Qubit and Bioanalyzer) and the pool will then be purified with paramagnetic beads to remove short and long fragments (<200 bp and >800 bp). After subsequent quality control (Qubit and Bioanalyzer), the sample will be sequenced with a read length of 250 bp in paired mode (2×250 bp) and with minimum output and quality defined in the description of Part 4. The sequencing output will be demultiplexed, and at the same time, sequencing adapters will be removed from the data (according to information from the client), and for each library, two FASTQ files containing forward and reverse read will be delivered to the client – ideally via an online platform.
Expected volume over 24 months – 44 pools.

LOT-0006
Part 5 – Paired sequencing of a library/pool 2×250 bp with a quantity of at least 12.5 Gb and an average quality of nucleobase identification for each individual read (sequence) of at least 30 or higher (Phred quality score Q30) for at least 60% of reads.
The sequencing includes basic quality control, e.g., measured on a Bioanalyzer or similar device, sequencing, and basic demultiplexing of the obtained data, if this step is relevant, without further bioinformatics processing or data evaluation, according to the description of the individual parts in point 1.2 of the tender documentation.
Sequencing + supplementary services: DNA or cDNA samples including one or more negative controls will be prepared in the client's laboratory. After delivery of these samples to the supplier, their quality will be checked and suitability for sequencing will be determined. Amplicons will be prepared using microbial DNA-free polymerase (e.g., TP HS DNA-free 2x Master Mix, Top-Bio) using primers specified by the client (e.g., 515-806, with delivery and quality of primers ensured by the supplier), indexed by appropriate methods (e.g., dual indexes Nextera XT), purified using columns or another suitable method, and the resulting pool will undergo quality control and, if necessary, be purified with paramagnetic beads to remove short fragments (<200 bp). The resulting pool will be sequenced in a separate or shared run, ensuring a minimum guaranteed output of 20 thousand pair-end reads per sample (i.e., 10 Mb/sample), with quality defined in the description of Part 5. The total required output is thus at least 1 Gb of sequencing data in total, provided there is completely balanced output for all samples, as the minimum required volume of sequencing data must be achieved for each individual sample. The sequencing output will be demultiplexed, and at the same time, sequencing adapters will be removed from the data, and for each library/amplicon, two FASTQ files containing forward and reverse read will be delivered to the client – ideally via an online platform.
Expected volume over 24 months – 7 pools.

LOT-0007
Part 6 – Paired sequencing of a library/pool 2×300 bp with a quantity of at least 6 Gb and an average quality of nucleobase identification for each individual read (sequence) of at least 30 or higher (Phred quality score Q30) for at least 50% of reads.
The sequencing includes basic quality control, e.g., measured on a Bioanalyzer or similar device, sequencing, and basic demultiplexing of the obtained data, if this step is relevant, without further bioinformatics processing or data evaluation, according to the description of the individual parts in point 1.2 of the tender documentation.
Sequencing + supplementary services: The supplied sequencing pool, which includes up to 384 individual libraries/amplicons double-indexed for example by Nextera XT barcodes, will be checked (Qubit and Bioanalyzer) and the pool will then be purified with paramagnetic beads to remove short fragments (e.g., <250 bp or <350 bp, according to the client’s requirements). After re-checking quality (Qubit and Bioanalyzer), the sample will be sequenced with a read length of 300 bp in paired mode (2×300 bp) with minimum output and quality defined in the description of Part 6. The sequencing output will then be demultiplexed, and at the same time, sequencing adapters will be removed from the data (according to information from the client), and for each library/amplicon, two FASTQ files containing forward and reverse read will be delivered to the client – ideally via an online platform.
Expected volume over 24 months – 13 pools.

LOT-0008
Part 7 – Paired sequencing of a library/pool 2×300 bp with a quantity of at least 15 Gb and an average quality of nucleobase identification for each individual read (sequence) of at least 30 or higher (Phred quality score Q30) for at least 50% of reads.
The sequencing includes basic quality control, e.g., measured on a Bioanalyzer or similar device, sequencing, and basic demultiplexing of the obtained data, if this step is relevant, without further bioinformatics processing or data evaluation, according to the description of the individual parts in point 1.2 of the tender documentation.
Sequencing + supplementary services: This example involves a contract for sequencing meta-transcriptomes, comprising 20 individual RNA samples isolated from soil in the client's laboratory. After delivery to the supplier, their quality will be checked and suitability for sequencing will be determined. Bacterial and fungal rRNA fragments will be removed from the samples by ribodepletion (only one type of rRNA may be removed, according to client requirements), residual DNA (using RNase-free DNase) will be removed, and the supplier will then prepare a pool of cDNA libraries for sequencing (sequencing pool) with unique indexes for each sample, fragmenting the DNA according to the client’s requirements (e.g., to 500 bp or 800 bp), or possibly without fragmentation. This pool will then be sequenced with a read length of 300 bp in paired mode (2×300 bp) with a minimum output of 15 Gb and quality defined in the description of Part 7. The sequencing output will then be demultiplexed, and at the same time, sequencing adapters will be removed from the data. For each library, two FASTQ files containing forward and reverse read will be delivered to the client – ideally via an online platform.
Expected volume over 24 months – 2 contracts.

LOT-0009
Part 8 – Long-read sequencing of minimum length 1500 bp with a quantity of at least 4 Gb and an average quality of nucleobase identification for each individual read (sequence) of at least 25 or higher (Phred quality score Q25) for 70% of reads.
The sequencing includes basic quality control, e.g., measured on a Bioanalyzer or similar device, sequencing, and basic demultiplexing of the obtained data, if this step is relevant, without further bioinformatics processing or data evaluation, according to the description of the individual parts in point 1.2 of the tender documentation.
Sequencing + supplementary services: This example involves sequencing total environmental DNA extracted from soil or wood samples, without further amplification of nucleic acids. After the delivery of a set of 8 DNA samples isolated in the client's laboratory, the supplier will check their quality and determine their suitability for sequencing. The samples will then be labeled with suitable barcodes to allow their distinction, and their pool will be sequenced using long-read methods with minimum output and quality defined in the description of Part 8. The sequencing output will then be demultiplexed, and at the same time, any sequencing adapters will be removed from the data (according to the sequencing platform used). For each library (sample), one FASTQ file containing sequences from the respective sample, without other sequences (either from other samples or sequencing controls), will be delivered to the client – ideally via an online platform.
Expected volume over 24 months – 12 sets.