RNA molecule RNA fragment Activity Intro Slide: When performing an RNA-seq experiment, we can make several changes to the sequencing protocol that can have drastic effects on our ability to align and analyze the resulting data. These include increasing the length of the RNA-seq reads, performing paired-end sequencing, or using fragment length information. Here, we’re going to explore each of these options and see how they affect our sequencing results. When performing an RNA-seq experiment, we need to prepare RNA molecules for sequencing. These include breaking large RNA molecules into smaller pieces (fragments),

RNA molecule RNA fragment Activity Intro Slide: When performing an RNA-seq experiment, we can make several changes to the sequencing protocol that can have drastic effects on our ability to align and analyze the resulting data. These include increasing the length of the RNA-seq reads, performing paired-end sequencing, or using fragment length information. Here, we’re going to explore each of these options and see how they affect our sequencing results. fragment ready for sequencing When performing an RNA-seq experiment, we need to prepare RNA molecules for sequencing. These include breaking large RNA molecules into smaller pieces (fragments), and making other changes to the molecule so it is recognized by the sequencing machine.

RNA molecule RNA fragment Activity Intro Slide: When performing an RNA-seq experiment, we can make several changes to the sequencing protocol that can have drastic effects on our ability to align and analyze the resulting data. These include increasing the length of the RNA-seq reads, performing paired-end sequencing, or using fragment length information. Here, we’re going to explore each of these options and see how they affect our sequencing results. fragment ready for sequencing Sequencing read (30 bp) cccaaccttcccacagctcacagcatcccc When performing an RNA-seq experiment, we need to prepare RNA molecules for sequencing. These include breaking large RNA molecules into smaller pieces (fragments), and making other changes to the fragments so they are recognized by the sequencing machine. Finally, we load the fragments into the sequencer which reads a specific number of letters (or base pairs) from one end of the fragment.

Paired-end sequencing RNA molecule RNA fragment Activity Intro Slide: When performing an RNA-seq experiment, we can make several changes to the sequencing protocol that can have drastic effects on our ability to align and analyze the resulting data. These include increasing the length of the RNA-seq reads, performing paired-end sequencing, or using fragment length information. Here, we’re going to explore each of these options and see how they affect our sequencing results. fragment ready for sequencing Sequencing read (30 bp) cccaaccttcccacagctcacagcatcccc Possible modifications: Increase read length Paired-end sequencing Fragment length info However, we can make several changes to the way we perform the sequencing. These changes can have a huge effect on our ability to align and analyze the resulting data. These include increasing the length of the RNA-seq reads, performing paired-end sequencing, or using fragment length information. Here, we’re going to explore each of these options and see how they affect our sequencing results.

RNA molecule RNA fragment Increase read length: When performing an RNA-seq experiment, we sequence a specific number of nucleotides from one end of an RNA fragment. We can chose to sequence more nucleotides, which reveals more of the RNA fragment’s sequence. Here we increased the read length from 30 bp to 75 bp. fragment ready for sequencing Sequencing read (30 bp) cccaaccttcccacagctcacagcatcccc OR Sequencing read (75 bp) cccaaccttcccacagctcacagcatccccccacagcatggacagcatgctgccctctggagaaggtggcccaaa Increase read length: When performing an RNA-seq experiment, we sequence a specific number of nucleotides from one end of an RNA fragment. We can chose to sequence more nucleotides, which reveals more of the RNA fragment’s sequence. Here we increased the read length from 30 bp to 75 bp.

Paired-end sequencing: RNA molecule RNA fragment Paired-end sequencing: In RNA-seq experiments, we generate sequencing reads from the ends of RNA fragments. We have the option of sequencing one, or both ends of an RNA fragment. Since we generate a pair of reads when we sequence both ends of a fragment, we call this method paired-end sequencing. fragment ready for sequencing Sequencing reads (paired ) cccaaccttcccacagctcacagcatcccc acaggataagagggtcatcgccttctagct Read Paired read Paired-end sequencing: In RNA-seq experiments, we generate sequencing reads from the ends of RNA fragments. We have the option of sequencing one, or both ends of an RNA fragment. Since we generate a pair of reads when we sequence both ends of a fragment, we call this method paired-end sequencing.

Use fragment length information: RNA molecule RNA fragment Use fragment length information: When preparing RNA fragments for sequencing, we can measure the average lengths of all the fragments in our sample. If we use this information when performing paired-end sequencing, we can calculate the distance we expect to see between read pairs. fragment ready for sequencing 200-300 bp Measure fragment length 50 bp 50 bp Sequencing reads (paired ) Calculate distance between reads 100-200 bp Use fragment length information: When preparing RNA fragments for sequencing, we can measure the average lengths of all the fragments in our sample. If we use this information when performing paired-end sequencing, we can calculate the distance we expect to see between read pairs.