What is the difference between a Read and a Fragment in RNA-seq?

In RNA-seq (and other sequencing technologies like whole-genome sequencing), the terms Read and Fragment are often used, and they have distinct meanings. Here’s a detailed explanation of the difference between the two, along with how they relate to single-end and paired-end sequencing:

1. Fragment

• A fragment refers to a physical piece of DNA or RNA that is generated during the library preparation process.
• In RNA-seq, RNA molecules are first converted into complementary DNA (cDNA) and then fragmented into smaller pieces. These pieces are called fragments.
• The size of these fragments is often controlled during library preparation and is referred to as the insert size (the length of the DNA/RNA between the sequencing adapters).
• Fragments are the templates for sequencing.

2. Read

• A read is the sequenced output of a fragment (or part of a fragment) generated by the sequencing machine.
• Each read corresponds to the sequence of nucleotides determined by the sequencer.
• Reads can be generated from one or both ends of a fragment, depending on the sequencing strategy (single-end or paired-end).

3. Single-End vs. Paired-End Sequencing

• Single-End Sequencing:
  • Only one end of the fragment is sequenced.
  • This produces one read per fragment.
  • Example: If a fragment is 300 bp long, and the read length is 100 bp, you will get a single 100 bp read from one end of the fragment.
• Paired-End Sequencing:
  • Both ends of the fragment are sequenced.
  • This produces two reads per fragment (one from each end).
  • Example: If a fragment is 300 bp long, and the read length is 100 bp, you will get two 100 bp reads: one from the forward end and one from the reverse end of the fragment.

4. Relationship Between Fragment and Read

• A fragment is the original piece of DNA/RNA, while a read is the sequenced output of that fragment.
• In single-end sequencing, one read corresponds to one end of a fragment.
• In paired-end sequencing, two reads correspond to the two ends of the same fragment.

5. Example in RNA-seq

• Suppose you have an RNA molecule that is converted into cDNA and fragmented into pieces of 300 bp.
• During sequencing:
  • In single-end mode, you might get a 100 bp read from one end of the fragment.
  • In paired-end mode, you might get two 100 bp reads: one from the forward end and one from the reverse end of the same fragment.

6. Why Does This Matter?

• Fragment Length vs. Read Length:
  • The fragment length (insert size) is the actual size of the DNA/RNA piece.
  • The read length is the number of bases sequenced from the fragment.
  • For example, a 300 bp fragment might produce two 100 bp reads in paired-end sequencing, leaving a 100 bp unsequenced gap in the middle.
• Applications:
  • Paired-end sequencing provides more information about the fragment, such as the distance between the two reads, which can help with alignment and structural variant detection.
  • Single-end sequencing is simpler and cheaper but provides less information.

7. Key Points

• A fragment is the physical piece of DNA/RNA being sequenced.
• A read is the sequenced output of a fragment.
• Single-end sequencing produces one read per fragment.
• Paired-end sequencing produces two reads per fragment (one from each end).

8. Visualization

Here’s a simple visualization:

Fragment:   ----------------------------- (300 bp)
Single-End: ---------- (100 bp read from one end)
Paired-End: ---------- (100 bp read from one end)
            ----------------------------- (300 bp)
            ---------- (100 bp read from the other end)

Understanding the difference between fragment and read is crucial for interpreting RNA-seq data, designing experiments, and analyzing sequencing results.