.2bit format (from the UCSC Genome Browser FAQ)

A .2bit file stores multiple DNA sequences (up to 4 Gb total) in a compact randomly-accessible format. The file contains masking information as well as the DNA itself.

The file begins with a 16-byte header containing the following fields:

• signature - the number 0x1A412743 in the architecture of the machine that created the file
• version - zero for now. Readers should abort if they see a version number higher than 0
• sequenceCount - the number of sequences in the file
• reserved - always zero for now

All fields are 32 bits unless noted. If the signature value is not as given, the reader program should byte-swap the signature and check if the swapped version matches. If so, all multiple-byte entities in the file will have to be byte-swapped. This enables these binary files to be used unchanged on different architectures.

The header is followed by a file index, which contains one entry for each sequence. Each index entry contains three fields:

• nameSize - a byte containing the length of the name field
• name - the sequence name itself (in ASCII-compatible byte string), of variable length depending on nameSize
• offset - the 32-bit offset of the sequence data relative to the start of the file, not aligned to any 4-byte padding boundary

The index is followed by the sequence records, which contain nine fields:

• dnaSize - number of bases of DNA in the sequence
• nBlockCount - the number of blocks of Ns in the file (representing unknown sequence)
• nBlockStarts - an array of length nBlockCount of 32 bit integers indicating the (0-based) starting position of a block of Ns
• nBlockSizes - an array of length nBlockCount of 32 bit integers indicating the length of a block of Ns
• maskBlockCount - the number of masked (lower-case) blocks
• maskBlockStarts - an array of length maskBlockCount of 32 bit integers indicating the (0-based) starting position of a masked block
• maskBlockSizes - an array of length maskBlockCount of 32 bit integers indicating the length of a masked block
• reserved - always zero for now
• packedDna - the DNA packed to two bits per base, represented as so: T - 00, C - 01, A - 10, G - 11. The first base is in the most significant 2-bit byte; the last base is in the least significant 2 bits. For example, the sequence TCAG is represented as 00011011.

In this format, it is neither possible nor necessary to store Ns in the main sequence, and one wouldn't expect them to take up space there. However, they do; hard masked sequence is typically stored as many Ts. The sensible way to treat these is probably to just say there are two kinds of implied annotation (repeats and large gaps for a typical genome), which can be interpreted in whatever way fits.