N50 is too small

[ericgonzalezs]

Hi,

I am running hifiasm 0.19.5-r592 like this:

          hifiasm -o Outfile -t 37 1.ccs.fastq.gz 2.ccs.fastq.gz

I don't have Hi-C data or parental information.

The end of the run looks like this

[M::ha_hist_line] 1: ****************************************************************************************************> 6553493 [M::ha_hist_line] 2: ****************************************************************************************************> 664815 [M::ha_hist_line] 3: *************************************************************************************************> 238583 [M::ha_hist_line] 4: ******************************************************************************* 138832 [M::ha_hist_line] 5: ***************************************************** 93274 [M::ha_hist_line] 6: ****************************************** 75104 [M::ha_hist_line] 7: *************************************** 68260 [M::ha_hist_line] 8: ************************************* 66072 [M::ha_hist_line] 9: ************************************* 65076 [M::ha_hist_line] 10: ************************************** 67037 [M::ha_hist_line] 11: *************************************** 68580 [M::ha_hist_line] 12: **************************************** 70875 [M::ha_hist_line] 13: ******************************************* 75145 [M::ha_hist_line] 14: ******************************************* 76452 [M::ha_hist_line] 15: ******************************************** 77232 [M::ha_hist_line] 16: ********************************************* 78982 [M::ha_hist_line] 17: ********************************************** 81545 [M::ha_hist_line] 18: ************************************************ 84328 [M::ha_hist_line] 19: ************************************************ 85683 [M::ha_hist_line] 20: *************************************************** 89871 [M::ha_hist_line] 21: **************************************************** 91582 [M::ha_hist_line] 22: ***************************************************** 93591 [M::ha_hist_line] 23: ****************************************************** 94630 [M::ha_hist_line] 24: ******************************************************* 97293 [M::ha_hist_line] 25: ******************************************************** 99060 [M::ha_hist_line] 26: ********************************************************* 100531 [M::ha_hist_line] 27: *********************************************************** 103554 [M::ha_hist_line] 28: ************************************************************ 105952 [M::ha_hist_line] 29: ************************************************************* 108301 [M::ha_hist_line] 30: **************************************************************** 113056 [M::ha_hist_line] 31: ***************************************************************** 114653 [M::ha_hist_line] 32: ****************************************************************** 117001 [M::ha_hist_line] 33: ******************************************************************** 119566 [M::ha_hist_line] 34: ********************************************************************** 124171 [M::ha_hist_line] 35: ************************************************************************ 127794 [M::ha_hist_line] 36: ************************************************************************** 131147 [M::ha_hist_line] 37: **************************************************************************** 134549 [M::ha_hist_line] 38: ****************************************************************************** 137141 [M::ha_hist_line] 39: ******************************************************************************** 141079 [M::ha_hist_line] 40: ********************************************************************************** 145386 [M::ha_hist_line] 41: ************************************************************************************ 149034 [M::ha_hist_line] 42: ************************************************************************************** 151559 [M::ha_hist_line] 43: **************************************************************************************** 156339 [M::ha_hist_line] 44: ****************************************************************************************** 159693 [M::ha_hist_line] 45: ******************************************************************************************** 162476 [M::ha_hist_line] 46: ********************************************************************************************* 165273 [M::ha_hist_line] 47: *********************************************************************************************** 168611 [M::ha_hist_line] 48: ************************************************************************************************* 171151 [M::ha_hist_line] 49: ************************************************************************************************** 173538 [M::ha_hist_line] 50: *************************************************************************************************** 174271 [M::ha_hist_line] 51: *************************************************************************************************** 175582 [M::ha_hist_line] 52: **************************************************************************************************** 176798 [M::ha_hist_line] 53: *************************************************************************************************** 175634 [M::ha_hist_line] 54: ************************************************************************************************** 173620 [M::ha_hist_line] 55: ************************************************************************************************ 170105 [M::ha_hist_line] 56: ********************************************************************************************** 166707 [M::ha_hist_line] 57: ********************************************************************************************* 164560 [M::ha_hist_line] 58: ******************************************************************************************* 160150 [M::ha_hist_line] 59: ***************************************************************************************** 156783 [M::ha_hist_line] 60: ************************************************************************************* 150268 [M::ha_hist_line] 61: *********************************************************************************** 147189 [M::ha_hist_line] 62: ********************************************************************************* 142439 [M::ha_hist_line] 63: ****************************************************************************** 138059 [M::ha_hist_line] 64: **************************************************************************** 133773 [M::ha_hist_line] 65: ************************************************************************* 128702 [M::ha_hist_line] 66: *********************************************************************** 124742 [M::ha_hist_line] 67: ******************************************************************** 119447 [M::ha_hist_line] 68: ***************************************************************** 115623 [M::ha_hist_line] 69: *************************************************************** 110814 [M::ha_hist_line] 70: ************************************************************ 106870 [M::ha_hist_line] 71: *********************************************************** 104929 [M::ha_hist_line] 72: ********************************************************** 102664 [M::ha_hist_line] 73: ********************************************************* 101022 [M::ha_hist_line] 74: ******************************************************** 99642 [M::ha_hist_line] 75: ******************************************************* 97516 [M::ha_hist_line] 76: ******************************************************* 97241 [M::ha_hist_line] 77: ****************************************************** 96319 [M::ha_hist_line] 78: ****************************************************** 94938 [M::ha_hist_line] 79: ****************************************************** 95249 [M::ha_hist_line] 80: ****************************************************** 94788 [M::ha_hist_line] 81: ***************************************************** 94251 [M::ha_hist_line] 82: ***************************************************** 94334 [M::ha_hist_line] 83: ****************************************************** 95353 [M::ha_hist_line] 84: ****************************************************** 96092 [M::ha_hist_line] 85: ******************************************************* 96984 [M::ha_hist_line] 86: ******************************************************* 97334 [M::ha_hist_line] 87: ******************************************************** 99408 [M::ha_hist_line] 88: ********************************************************* 100278 [M::ha_hist_line] 89: ********************************************************* 101335 [M::ha_hist_line] 90: ********************************************************** 102159 [M::ha_hist_line] 91: ********************************************************* 101386 [M::ha_hist_line] 92: *********************************************************** 104333 [M::ha_hist_line] 93: *********************************************************** 104884 [M::ha_hist_line] 94: ************************************************************ 105745 [M::ha_hist_line] 95: *********************************************************** 104773 [M::ha_hist_line] 96: *********************************************************** 105148 [M::ha_hist_line] 97: *********************************************************** 105038 [M::ha_hist_line] 98: *********************************************************** 104853 [M::ha_hist_line] 99: ************************************************************ 105245 [M::ha_hist_line] 100: ************************************************************* 107025 [M::ha_hist_line] 101: ************************************************************ 105308 [M::ha_hist_line] 102: *********************************************************** 104599 [M::ha_hist_line] 103: *********************************************************** 105094 [M::ha_hist_line] 104: *********************************************************** 104954 [M::ha_hist_line] 105: ********************************************************** 102855 [M::ha_hist_line] 106: ********************************************************* 101144 [M::ha_hist_line] 107: ********************************************************* 100616 [M::ha_hist_line] 108: ******************************************************** 99452 [M::ha_hist_line] 109: ******************************************************* 98089 [M::ha_hist_line] 110: ****************************************************** 96040 [M::ha_hist_line] 111: ***************************************************** 94309 [M::ha_hist_line] 112: *************************************************** 90903 [M::ha_hist_line] 113: ************************************************** 88228 [M::ha_hist_line] 114: ************************************************* 85892 [M::ha_hist_line] 115: *********************************************** 82759 [M::ha_hist_line] 116: ********************************************* 79511 [M::ha_hist_line] 117: ******************************************** 77894 [M::ha_hist_line] 118: ****************************************** 74716 [M::ha_hist_line] 119: ***************************************** 71860 [M::ha_hist_line] 120: *************************************** 68861 [M::ha_hist_line] 121: ************************************* 64805 [M::ha_hist_line] 122: *********************************** 62098 [M::ha_hist_line] 123: ********************************* 59022 [M::ha_hist_line] 124: ******************************** 55884 [M::ha_hist_line] 125: ***************************** 52090 [M::ha_hist_line] 126: **************************** 49896 [M::ha_hist_line] 127: ************************** 46370 [M::ha_hist_line] 128: ************************* 43701 [M::ha_hist_line] 129: ************************ 41741 [M::ha_hist_line] 130: ********************** 38372 [M::ha_hist_line] 131: ******************** 35939 [M::ha_hist_line] 132: ******************* 33080 [M::ha_hist_line] 133: ****************** 31038 [M::ha_hist_line] 134: **************** 28588 [M::ha_hist_line] 135: *************** 26640 [M::ha_hist_line] 136: ************** 24424 [M::ha_hist_line] 137: ************* 22264 [M::ha_hist_line] 138: *********** 20289 [M::ha_hist_line] 139: *********** 18646 [M::ha_hist_line] 140: ********** 17149 [M::ha_hist_line] 141: ********* 15911 [M::ha_hist_line] 142: ******** 14123 [M::ha_hist_line] 143: ******* 12757 [M::ha_hist_line] 144: ******* 11887 [M::ha_hist_line] 145: ****** 10624 [M::ha_hist_line] 146: ***** 9720 [M::ha_hist_line] 147: ***** 9223 [M::ha_hist_line] 148: ***** 8389 [M::ha_hist_line] 149: ***** 8038 [M::ha_hist_line] 150: **** 7455 [M::ha_hist_line] 151: **** 7047 [M::ha_hist_line] 152: **** 6430 [M::ha_hist_line] 153: *** 5799 [M::ha_hist_line] 154: *** 5563 [M::ha_hist_line] 155: *** 5333 [M::ha_hist_line] 156: *** 5142 [M::ha_hist_line] 157: *** 4785 [M::ha_hist_line] 158: ** 4342 [M::ha_hist_line] 159: ** 4306 [M::ha_hist_line] 160: ** 4047 [M::ha_hist_line] 161: ** 3943 [M::ha_hist_line] 162: ** 3832 [M::ha_hist_line] 163: ** 3755 [M::ha_hist_line] 164: ** 3758 [M::ha_hist_line] 165: ** 3753 [M::ha_hist_line] 166: ** 3525 [M::ha_hist_line] 167: ** 3397 [M::ha_hist_line] 168: ** 3164 [M::ha_hist_line] 169: ** 3195 [M::ha_hist_line] 170: ** 3194 [M::ha_hist_line] 171: ** 3097 [M::ha_hist_line] 172: ** 3048 [M::ha_hist_line] 173: ** 2915 [M::ha_hist_line] 174: ** 2916 [M::ha_hist_line] 175: ** 2759 [M::ha_hist_line] 176: ** 2826 [M::ha_hist_line] 177: ** 2952 [M::ha_hist_line] 178: ** 2804 [M::ha_hist_line] 179: ** 2780 [M::ha_hist_line] 180: ** 2931 [M::ha_hist_line] 181: ** 2821 [M::ha_hist_line] 182: ** 2851 [M::ha_hist_line] 183: ** 2753 [M::ha_hist_line] 184: ** 2710 [M::ha_hist_line] 185: * 2635 [M::ha_hist_line] 186: * 2606 [M::ha_hist_line] 187: * 2584 [M::ha_hist_line] 188: * 2479 [M::ha_hist_line] 189: * 2519 [M::ha_hist_line] 190: * 2435 [M::ha_hist_line] 191: * 2499 [M::ha_hist_line] 192: * 2435 [M::ha_hist_line] 193: * 2441 [M::ha_hist_line] 194: * 2464 [M::ha_hist_line] 195: * 2391 [M::ha_hist_line] 196: * 2387 [M::ha_hist_line] 197: * 2293 [M::ha_hist_line] 198: * 2316 [M::ha_hist_line] 199: * 2254 [M::ha_hist_line] 200: * 2314 [M::ha_hist_line] 201: * 2257 [M::ha_hist_line] 202: * 2104 [M::ha_hist_line] 203: * 2193 [M::ha_hist_line] 204: * 1967 [M::ha_hist_line] 205: * 2084 [M::ha_hist_line] 206: * 2037 [M::ha_hist_line] 207: * 2015 [M::ha_hist_line] 208: * 2056 [M::ha_hist_line] 209: * 1934 [M::ha_hist_line] 210: * 2023 [M::ha_hist_line] 211: * 1945 [M::ha_hist_line] 212: * 1931 [M::ha_hist_line] 213: * 1941 [M::ha_hist_line] 214: * 1887 [M::ha_hist_line] 215: * 1946 [M::ha_hist_line] 216: * 1989 [M::ha_hist_line] 217: * 1973 [M::ha_hist_line] 218: * 1951 [M::ha_hist_line] 219: * 1926 [M::ha_hist_line] 220: * 1977 [M::ha_hist_line] 221: * 1705 [M::ha_hist_line] 222: * 1843 [M::ha_hist_line] 223: * 1822 [M::ha_hist_line] 224: * 1833 [M::ha_hist_line] 225: * 1746 [M::ha_hist_line] 226: * 1684 [M::ha_hist_line] 227: * 1573 [M::ha_hist_line] 228: * 1726 [M::ha_hist_line] 229: * 1651 [M::ha_hist_line] 230: * 1601 [M::ha_hist_line] 231: * 1605 [M::ha_hist_line] 232: * 1575 [M::ha_hist_line] 233: * 1601 [M::ha_hist_line] 234: * 1632 [M::ha_hist_line] 235: * 1562 [M::ha_hist_line] 236: * 1519 [M::ha_hist_line] 237: * 1517 [M::ha_hist_line] 238: * 1466 [M::ha_hist_line] 239: * 1540 [M::ha_hist_line] 240: * 1542 [M::ha_hist_line] 241: * 1575 [M::ha_hist_line] 242: * 1519 [M::ha_hist_line] 243: * 1528 [M::ha_hist_line] 244: * 1467 [M::ha_hist_line] 245: * 1356 [M::ha_hist_line] 246: * 1343 [M::ha_hist_line] 247: * 1377 [M::ha_hist_line] 248: * 1422 [M::ha_hist_line] 249: * 1242 [M::ha_hist_line] 250: * 1237 [M::ha_hist_line] 251: * 1231 [M::ha_hist_line] 252: * 1319 [M::ha_hist_line] 253: * 1345 [M::ha_hist_line] 254: * 1287 [M::ha_hist_line] 255: * 1259 [M::ha_hist_line] 256: * 1279 [M::ha_hist_line] 257: * 1121 [M::ha_hist_line] 258: * 1175 [M::ha_hist_line] 259: * 1150 [M::ha_hist_line] 260: * 1086 [M::ha_hist_line] 261: * 974 [M::ha_hist_line] 262: * 1143 [M::ha_hist_line] 263: * 1043 [M::ha_hist_line] 264: * 1067 [M::ha_hist_line] 265: * 1032 [M::ha_hist_line] 266: * 1069 [M::ha_hist_line] 267: * 1053 [M::ha_hist_line] 268: * 1023 [M::ha_hist_line] 269: * 974 [M::ha_hist_line] 270: * 974 [M::ha_hist_line] 271: * 962 [M::ha_hist_line] 272: * 927 [M::ha_hist_line] rest: *************************************************************** 110570 [M::ha_analyze_count] left: none [M::ha_analyze_count] right: count[100] = 107025 [M::ha_pt_gen] peak_hom: 100; peak_het: 52 [M::ha_ct_shrink::11950.14434.15] ==> counted 15248639 distinct minimizer k-mers [M::ha_pt_gen::] counting in normal mode [M::yak_count] collected 1015930235 minimizers [M::yak_count] collected 0 minimizers [M::ha_pt_gen::12037.38034.07] ==> indexed 1009376742 positions, counted 15248639 distinct minimizer k-mers [M::ha_assemble::12857.554[email protected]] ==> found overlaps for the final round [M::ha_print_ovlp_stat] # overlaps: 431100705 [M::ha_print_ovlp_stat] # strong overlaps: 229935566 [M::ha_print_ovlp_stat] # weak overlaps: 201165139 [M::ha_print_ovlp_stat] # exact overlaps: 415891167 [M::ha_print_ovlp_stat] # inexact overlaps: 15209538 [M::ha_print_ovlp_stat] # overlaps without large indels: 429908817 [M::ha_print_ovlp_stat] # reverse overlaps: 238262678 [M::ha_opt_update_cov_min] updated max_n_chain to 500 Writing reads to disk... Reads has been written. Writing ma_hit_ts to disk... ma_hit_ts has been written. Writing ma_hit_ts to disk... ma_hit_ts has been written. bin files have been written. [M::purge_dups] homozygous read coverage threshold: 100 [M::purge_dups] purge duplication coverage threshold: 125
[M::ug_ext_gfa::] # tips::719 Writing raw unitig GFA to disk... [M::ug_ext_gfa::] # tips::1 Writing processed unitig GFA to disk... [M::purge_dups] homozygous read coverage threshold: 100 [M::purge_dups] purge duplication coverage threshold: 125 [M::mc_solve:: # edges: 2180] [M::mc_solve_core_adv::0.221] ==> Partition [M::adjust_utg_by_primary] primary contig coverage range: [85, infinity] Writing KELP_RS252_RS253.bp.p_ctg.gfa to disk... [M::reduce_hamming_error_adv::0.450] # inserted edges: 446, # fixed bubbles: 5 [M::adjust_utg_by_trio] primary contig coverage range: [85, infinity] [M::recall_arcs] # transitive arcs::80 [M::recall_arcs] # new arcs::13336, # old arcs::10292 [M::clean_trio_untig_graph] # adjusted arcs::0 [M::adjust_utg_by_trio] primary contig coverage range: [85, infinity] [M::recall_arcs] # transitive arcs::92 [M::recall_arcs] # new arcs::13498, # old arcs::10534 [M::clean_trio_untig_graph] # adjusted arcs::0 [M::output_trio_graph_joint] dedup_base::23888871, miss_base::0 Writing KELP_RS252_RS253.bp.hap1.p_ctg.gfa to disk... Writing KELP_RS252_RS253.bp.hap2.p_ctg.gfa to disk... Inconsistency threshold for low-quality regions in BED files: 70% [M::main] Version: 0.19.5-r592 [M::main] CMD: hifiasm -o Outfile -t 37 1.ccs.fastq.gz 2.ccs.fastq.gz [M::main] Real time: 14786.619 sec; CPU: 442384.487 sec; Peak RSS: 72.014 GB

The coverage we have with Hifi data is quite high, we have ~60X. However, the median length of the reads is ~9Kb. With many reads around 5kb and they look like this image for one of the libraries:

The kmer plot from Genomescope for one of the libraries looks like this:

The other library is very similar in length and for genomescope.

In the end, the N50 is quite small 1.74 Mb with a total of 514.79 Mb assembled (taking into account the primary assembly). The expected size of the genome is 588Mb with ~32 Chromosomes. I was expecting a Bigger N50 and more bp in the assembly. I read that to improve the continuity of the assembly I could increase the values for -N and -D and I read in another issue that your recommendations for -N were 20 and for -D 400. Do you recommend the same values for this data? What else could you recommend me to improve this assembly? Do you think the size of the reads is affecting this assembly?

Many thanks